HEPATIC METABOLISM DURING SEPSIS Paulo Roberto Leitao de

Transcrição

HEPATIC METABOLISM DURING SEPSIS
A thesis presented in part fufilment of the
the requirements for the degree
of
Doctor of Philosophy
of the
University of Oxford
by
Paulo Roberto Leitao de Vasconcelos
Trinity Term
1987
Green College
Oxford
This thesis is dedicated to my wife Dione,
to my daughter Raquel,
to my sons Paulo Roberto and Marcelo,
and
to my parents
Acknowledgements
I am extremely grateful to both my biochemical and clinical supervisors.
I will always be indebted to Dr. Williamson, who made possible the realization of
the experimental work, when, in his kind way, he allowed me to work in the Metabolic
Research Laboratory. I would consider myself very gifted if I could keep with me a tiny
part of Dr Williamson's insight into scientific thinking. I am also very grateful for his
constant encouragement, sense of humour, and skilled supervision, both at the bench
throughout the project, and also during the period of writing.
I am profoundly grateful to Mr. Kettlewell for all his guidance during the clinical
studies. He accepted me to do research in Oxford and has always been a fund of
encouragement, enthusiasm, and constructive ideas, as well as an example of a surgeon
concerned with the day to day metabolic status of his patients. I also would like to thank
him for having taught me how to clinically assess and parenterally feed a surgical patient
in need, and to ask many questions about this unphysiological, but sometimes life-saving,
form of nutrition. Mr. Kettlewell has always made sharp scientific criticisms during his
supervision of the clinical and written work.
I would like to thank Mrs. Vera Ilic for introducing me into the world of enzymatic
analysis, and for doing so many insulin assays. I am grateful to Mr. Reginald Hems for
teaching me the art of preparing isolated hepatocytes. I am also grateful to Dr. Geoffrey
Gibbons for his supervision and assitance in the studies involving cholesterol
metabolism. My thanks to Mrs. Claudia Oller do Nascimento who has very kindly
performed the lipoprotein lipase assays.
I am very grateful to Miss Jane Clarke and Mr. Mike Dixon for helping me with the
clinical studies. Ms. Mellany Burnnet from the Diabetes Research Laboratory has kindly
measured the plasma insulin from parenterally fed patients. I also would like to express my
thanks to the consultant surgeons of the John Radcliffe Hospital who allowed me to study
their patients, and mostly to the patients who agreed to be involved in the parenteral
nutrition studies.
I am sincerely indebted to C.A.P.E.S. (Brazil) for having granted me with their
scholarship, and for giving support to this project.
I would like to thank Dr. Alexandre Kalache and Dr. Haroldo Juaçaba, in Brazil, for
all their encouragement and support about my decision to come to Oxford. I would like to
acknowledge skillful computer assistance from Dr. Manoel Odorico Moraes Filho. I am
very grateful to Dr. Richard Dawking for allowing me to use his laser printer.
Finally, I would like to thank my wife Dione, who has been so supportive and
understanding all over these four years, always encouraging me to persevere.
HEPATIC METABOLISM DURING SEPSIS
Thesis submitted to the University of Oxford for the
Degree of Doctor of Philosophy
Paulo R. L. de Vasconcelos
Green College
Trinity Term, 1987
Oxford
Abstract
Sepsis is an important clinical problem causing morbidity and mortality. Survival
depends on the metabolic adaptation, which is part of a concerted physiological and
immunological response. Time, nutritional status, the hormonal milieu, and the severity of
infection modulate the metabolic response. The liver has a central role in intermediary metabolism
during sepsis.
The time-course (12, 24, and 48h) of changes in hepatic metabolism during sepsis was
studied. A rat peritonitis model, which mimics human abdominal sepsis, was used. Moderate
sepsis was induced by caecal-ligation-and- puncture.
Blood and hepatic metabolite concentrations, and gluconeogenesis in isolatedhepatocytes were measured. Sepsis increased blood and liver gluconeogenic precursors, but
caused no change in blood glucose or plasma non-esterified fatty-acids; blood and hepatic ketonebodies decreased. These changes were accompanied by increased plasma insulin in septic-rats.
Gluconeogenesis from all precursors (alanine, lactate, pyruvate, glutamine, dihydroxyacetone)
was decreased by sepsis (48h).
Sepsis impaired ketogenesis from long-chain fatty-acids (oleate) in hepatocytes from
septic rats at all
time-points, whereas
when
short-chain fatty-acids (butyrate) were the
precursors ketogenesis was unchanged. This may be due to decreased entry of long-chain acylCoA into the mitochondria in the liver of septic-rats.
Sepsis stimulated in vivo hepatic lipogenesis (24, 48h) and cholesterogenesis (12, 24,
48h).
The
increased
cholesterogenesis
was
accompanied
by
increase
in
total
hydroxymethylglutaryl-CoA reductase activity, but no change in the active form of the enzyme
was observed.
The key factor responsible for these metabolic changes may be the elevated plasma
insulin concentrations. Treatment of septic-rats, with mannoheptulose or streptozotocin, to lower
their plasma insulin reversed many of the metabolic changes to the profile seen in control-rats,
supporting the view that hyperinsulinaemia is an important regulatory-factor in hepatic
metabolism during sepsis.
The hepatic metabolic changes induced by sepsis were compared to those found in
parenterally-fed patients. Many metabolic changes were common, and the hyperinsulinaemia seen
in both sepsis and total-parenteral-nutrition was again implicated.
TABLE OF CONTENTS
CHAPTER 1
Page
Introduction
01
Factors determining the development of infection
02
Factors increasing susceptibility to infection
04
The relation of infection and injury with malnutrition
05
and immunity
Historical Aspects and the importance of infection
09
The metabolic effects of starvation and injury
12
General description of the metabolic response to sepsis
15
Multiple organ failure
19
Sepsis and endotoxin
22
The effect of gram-positive bacterial components during
24
infection
The role of the phagocytic cells in infection
25
The role of the macrophage during sepsis
26
The role of interleukin-1 in the metabolic response to
30
sepsis
The importance of cachectin during sepsis
32
The role of corticosteroids in the response to sepsis
34
The counter-regulatory hormones and the regulation of
37
the metabolic response to sepsis
Sepsis and the effects of insulin
38
Biological response modifiers during sepsis
40
Objective of the study
45
TABLE OF CONTENTS
CHAPTER 2
Materials and Methods
Page
Biochemicals and Enzymes
47
Animals
47
Sepsis induced by caecal-ligation and puncture
47
Sham-operated rats
49
Bile duct-ligated rats
50
Preparation of blood, plasma and tisue samples
51
Determination of metabolites
52
Calculations of blood and liver metabolites
66
Insulin determination
66
Measurements of lipogenesis and cholesterogenesis
67
Preparation of isolated hepatocytes
69
Incubation procedure
72
Preparation of microsomes
74
Determination of hepatic enzyme activity
74
Determination of lipoprotein lipase activity
78
Measurements of radioactivity
79
Statistical Analysis
80
TABLE OF CONTENTS
Time course of changes in blood and liver
metabolites and plasma insulin after induction of sepsis
Page
CHAPTER 3
Introduction
81
Changes in blood and plasma metabolites
81
Changes in metabolite concentrations in the liver
83
Aims of the study
84
Results 85
Time course of changes in the concentrations of blood glucose,
85
lactate, pyruvate and alanine
Time course of changes in plasma insulin concentration
87
Time course of changes in plasma triacylglycerols and non-
88
esterified fatty acid concentrations
Time course of changes in the concentrations of blood ketone
89
bodies
Changes with time in the hepatic concentration of lactate,
90
pyruvate, alanine, glutamine and glutamate
Time course of changes in hepatic concentrations of glucose
93
Changes in glycogen concentration in the liver after induction
93
of sepsis
Time course of changes in the concentrations of hepatic
94
gluconeogenic intermediates in vivo
Time course of changes in hepatic concentrations of ketone
96
bodies in vivo
Time course of changes in hepatic concentrations of acetyl-CoA
97
and ATP in vivo
Discussion
98
TABLE OF CONTENTS
Time course of changes in rates of gluconeogenesis in
isolated hepatocytes during the induction of sepsis
CHAPTER 4
Page
Introduction
102
Design of the study
105
Results
106
Discussion
112
TABLE OF CONTENTS
Time course of changes in the metabolism of [1-14C]oleate
and
the rate of ketogenesis in isolated liver cells
after induction of sepsis
Page
CHAPTER 5
Introduction
116
Metabolism of long- and short-chain fatty acids
116
Changes in plasma non-esterified fatty acids and blood
118
hepatic ketone body concentration during sepsis
Design of the studies
Results
120
121
Time course of rates of ketogenesis from long- and short-
121
chain fatty acids in isolated hepatocytes
The metabolism of [1-14C]oleate during sepsis
123
Discussion
125
TABLE OF CONTENTS
Time course of changes in rates of lipogenesis and hepatic
cholesterogenesis in vivo after the induction of sepsis
CHAPTER 6
Page
Introduction
129
Experimental design
134
Results
135
Effects of sepsis on hepatic lipogenesis
135
Effects of sepsis on squalene and cholesterol synthesis
137
in vivo and hydroxymethylglutaryl-CoA reductase
activity in the liver
Effects of sepsis on lipoprotein lipase activity and rates of
140
lipogenesis in vivo in adipose tissue
The effect of the complete interruption of the entero-
142
hepatic circulation of bile acids accompainied by
sepsis on the rates of cholesterol and fatty acid
synthesis in the liver in vivo
Discussion
143
TABLE OF CONTENTS
The effects of modulation of plasma insulin on hepatic
metabolism during the induction of sepsis
CHAPTER 7
Page
Introduction
147
Experimental design
150
Results
151
The effects of mannoheptulose or streptozotocin treatment
151
on changes in concentrations of blood glucose, lactate,
pyruvate and alanine during the induction of sepsis
154
on the concentrations of blood ketone bodies during the
induction of sepsis
155
on plasma insulin concentration during the induction
of sepsis
156
on hepatic concentration of lactate, pyruvate, alanine
and glucose during the induction of sepsis
158
on the hepatic concentraton of metabolites involved
in gluconeogenesis during the induction of sepsis
163
on the changes in hepatic concentrations of ketone
bodies, acetyl-CoA and ATP during the induction of sepsis
165
on the changes in rates of lipogenesis and
cholesterogenesis in vivo during the induction of sepsis
The effects of mannoheptulose treatment on the metabolic
169
changes of sham-operated rats
The effects of insulin replacement, given to septic rats treated
172
with mannoheptulose, on the metabolic changes induced
by sepsis
Discussion
174
TABLE OF CONTENTS
General discussion
CHAPTER 8
Page
General discussion
181
The regulation of the metabolic response to sepsis
182
Is the metabolic response to sepsis adaptive?
184
Manipulation of the metabolic response to sepsis
185
TABLE OF CONTENTS
Common features of the changes in hepatic metabolism
induced by sepsis and by total parenteral nutrition
CHAPTER 9
Page
Introduction
189
Cyclical Parenteral Nutrition
192
Ezperimental Design
197
Study on liver dysfunction developing in parenterally fed
197
patients during sepsis
Randomized controlled study on standard versus tailored total
199
parenteral nutrition (TPN)
Controlled study on continuous versus cyclical total parenteral
203
nutrition (TPN)
Statistical analysis
206
Results
207
The effects of sepsis on liver function tests in patients
207
receiving total parenteral nutrition (TPN)
Tailored glucose versus standard glucose TPN regimen
210
Standard versus tailored TPN - Effect on nitrogen balance
212
Continuous versus cyclical TPN - Changes in blood metabolites
213
and plasma insulin over 24 h
Changes in liver function during standard versus tailored total
parenteral nutrition (TPN)
222
Changes in liver function during continuous versus cyclical
224
total parenteral nutrition (TPN)
Discussion
225
TABLE OF CONTENTS
Page
References
232
Publications arising from this Thesis
267
LIST OF FIGURES
Page
Fig. 1.1.
Changes in the determinats of sepsis
03
Fig. 1.2.
The relationship between sepsis/injury and malnutrition
06
Fig. 1.3.
Summary of the metabolic changes in starvation and injury
12
Fig. 1.4.
Phases of the metabolic response to injury/sepsis
14
Fig. 1.5.
Metabolic changes in response to sepsis
18
Fig. 1.6.
Some clinical aspects of sepsis and endotoxin treament
23
Fig. 1.7.
Role of the macrophage in initiating the immunological
28
response to sepsis
Fig. 2.1.
Caecal-ligation
48
Fig. 2.2.
Caecal-ligation and puncture
48
Fig. 2.3.
Sham-operation
50
Fig. 2.4.
Liver perfusion apparatus for hepatocyte isolation
71
Fig. 3.1.
Blood glucose
85
Fig. 3.2.
Blood lactate
86
Fig. 3.3.
Blood pyruvate
86
Fig. 3.4.
Blood alanine
87
Fig. 3.5.
Insulin concentration
88
Fig. 3.6.
Plasma triacylglycerol concentration
88
Fig. 3.7.
Non-esterified fatty acids
89
Fig. 3.8.
Blood ketone bodies
89
Fig. 3.9.
Liver lactate
90
Fig. 3.10.
Liver pyruvate
91
Fig. 3.11.
Liver alanine
91
Fig. 3.12.
Liver glutamine concentration
92
Fig. 3.13.
Hepatic glutamate concentration
92
Fig. 3.14.
Liver glucose
93
Fig. 3.15.
Metabolites involved in hepatic gluconeogenesis (24h)
95
Fig. 3.16.
Metabolites involved in hepatic gluconeogenesis (48h)
95
Fig. 3.17.
Liver ketone bodies
96
Fig. 3.18.
Acetyl-CoA concentration in the liver
97
Fig. 3.19.
ATP concentration in the liver
97
Fig. 4.1.
Gluconeogenesis and glycolysis
103
Fig. 4.2.
Design of the study (Chapter 4)
105
Fig. 4.3.
Rate of gluconeogenesis - no substrate
106
Fig. 4.4
Rate of gluconeogenesis from lactate [5mM]
107
LIST OF FIGURES
Page
Fig. 4.5.
Rates of gluconeogenesis from lactate
108
Fig. 4.6.
Rate of gluconeogenesis from alanine [5mM]
108
Fig. 4.7.
Rate of gluconeogenesis from pyruvate [5mM]
109
Fig. 4.8.
Rate of gluconeogenesis from glutamine [5mM]
110
Fig. 4.9.
Rate of gluconeogenesis from dihydroxyacetone [5mM]
111
Fig. 5.1.
Simplified scheme on the hepatic metabolism of long- and
117
shor-chain fatty acids
Fig. 5.2.
Design of the studies (Chapter 5)
120
Fig. 5.3.
Rate of ketone body formation - no added substrate
121
Fig. 5.4.
Rate of ketogenesis from butyrate [5mM]
122
Fig. 5.5
Rate of ketogenesis from oleate [2mM]
122
Fig. 5.6.
Metabolism of [1-14C]oleate
124
Fig. 6.1.
Simplified scheme of lipid metabolism in the liver
131
Fig. 6.2.
134
Fig. 6.7.
Experimental design (Chapter 6)
Rate of 3H2O incorporation into fatty acids
Rate of 3H2O incorporation into non-saponifiable lipids
Rate of 3H2O incorporation into lipids
Rate of 3H2O incorporation into squalene
Rate of 3H2O incorporation into cholesterol
Fig. 6.9.
HMG-CoA reductase activity
139
Fig. 6.10.
Rate of lipogenesis in white adipose tissue
140
Fig. 6.11.
Rate of lipogenesis in brown adipose tissue
141
Fig. 6.12.
Hepatic cholesterol synthesis and bile duct ligation
142
Fig. 7.1.
D-mannoheptulose
149
Fig. 6.3.
Fig. 6.4.
Fig. 6.5.
Fig. 6.6.
135
136
136
137
138
Fig. 7.2.
Experimental design (Chapter 7)
150
Fig. 7.3.
Blood glucose concentration
151
Fig. 7.4.
Blood lactate concentration
152
Fig. 7.5.
Blood pyruvate concentration
153
Fig. 7.6.
Blood alanine concentration
153
Fig. 7.7.
Blood concentration of ketone bodies
154
Fig. 7.8.
Plasma insulin concentration
155
Fig. 7.9.
Hepatic lactate concentration
156
Fig. 7.10.
Hepatic pyruvate concentration
157
Fig. 7.11.
Hepatic alanine concentration
157
Fig. 7.12.
Hepatic glucose concentration
158
Fig. 7.13.
Metabolites involved in gluconeogenesis
160
LIST OF FIGURES
Page
Fig. 7.14.
161
Fig. 7.15.
162
Fig. 7.16.
Hepatic concentration of ketone bodies
163
Fig. 7.17.
Hepatic concentration of acetyl-CoA
164
Fig. 7.18.
Fig. 7.19.
164
165
Fig. 7.22.
Hepatic concentration of ATP
Rate of 3H2O incorporation into non-saponifiable lipids
Rate of 3H2O incorporation into squalene
167
Fig. 7.23.
Fig. 7.24.
168
171
Fig. 7.25.
171
Fig. 7.26.
Metabolic changes in septic rats after treatment to lower
174
Fig. 7.20.
Fig. 7.21.
166
167
plasma insulin
Fig. 7.27.
Possible effects of sepsis and treatment to lower plasma
insulin on the hepatic glucose 6-phosphate/glucose
cycle
177
Fig. 8.1.
Possible changes in the metabolic response to sepsis
182
determined in part by the severity of infection
Fig. 8.2.
Possible cascade of events leading to the hepatic metabolic
186
response to sepsis
Fig. 9.1.
Some common metabolic features shared by sepsis and TPN
189
Fig. 9.2.
Criteria for selection of patients to receive cyclical TPN
193
Fig. 9.4.
Principal indications for home TPN
194
Fig. 9.5.
Continuous versus Cyclical TPN - Study design
203
Fig. 9.6.
Patients with abnormal albumin during sepsis and TPN
207
Fig. 9.7.
Patients with abnormal bilirubin during sepsis and TPN
Fig. 9.8.
Patients with abnormal alkaline phosphatase during
208
sepsis and TPN
Fig. 9.9.
Patients with abnormal AST during sepsis and TPN
209
Fig. 9.10.
Standard versus tailored TPN - Blood metabolites
211
Fig. 9.11.
Standard versus tailored TPN - Blood metabolites
211
Fig. 9.12.
Standard versus tailored TPN - Blood ketone bodies
212
Fig. 9.13.
Tailored versus standard TPN - Effect on nitrogen balance
213
Fig. 9.14.
Blood glucose
213
Fig. 9.15.
Blood pyruvate
214
LIST OF FIGURES
Page
Fig. 9.16.
Blood glutamate
215
Fig. 9.17.
Blood lactate
215
Fig. 9.18.
Blood alanine
216
Fig. 9.19.
Plasma insulin
216
Fig. 9.20.
Blood ketone bodies
217
Fig. 9.21.
Effects of cyclical TPN on blood pyruvate
218
Fig. 9.22.
Effects of cyclical TPN on blood glutamate
218
Fig. 9.23.
Effects of cyclical TPN on blood lactate
219
Fig. 9.24.
Effects of cyclical TPN on blood alanine
219
Fig. 9.25.
Effects of cyclical TPN on blood glucose
220
Fig. 9.26.
Effects of cyclical TPN on plasma insulin
220
Fig. 9.27.
Effects of cyclical TPN on blood ketone bodies
221
Fig. 9.28.
Continuous versus cyclical TPN - Effect on nitrogen balance
221
Fig. 9.29.
Changes with time in albumin during TPN
222
Fig. 9.30.
Changes with time in bilirubin during TPN
222
Fig. 9.31.
Changes with time in alkaline phosphatase during TPN
223
Fig. 9.32.
Changes with time in aspartate transaminase during TPN
223
List of Tables
Page
Table 3.1.
Time course of changes in the concentration of hepatic
94
gluconeogenic intermediated in vivo
Table 5.1.
Effects of sepsis on metabolism of [1-14C]oleate in isolated
124
hepatocytes from 48 h starved rats
Table 7.1.
159
on the hepatic concentration of metabolites involved
in gluconeogenesis after the induction of sepsis invivo
Table 7.2.
The effects of mannoheptulose treatment on the metabolic
170
changes of sham-operated rats in vivo
Table 7.3.
Th e effects of insulin administration on the metabolic
173
changes of septic rats treated with mannoheptulose
in vivo at 48 h after the operation
Table 9.1.
Patients fed on tailored or standard TPN regimen
200
Table 9.2.
Main indication for TPN
200
Table 9.3.
The Harris-Benedict equation
201
Table 9.4.
Patients who received cyclical parenteral nutrition
201
Table 9.5.
Nitrogen and energy intake
210
Table 9.6.
Nitrogen and energy according to the body weight
210
Table 9.7.
Tailored versus Standard TPN - Effects on liver function
224
tests
Table 9.8.
Continuous versus cyclical TPN - Effects on liver function
Table 7.9.
A comparison between blood metabolite values obtained
during the 'post-absorptive' cyclical TPN ("OFF" or
infusion free period) and those found in normal
controls after an overnight fast
224
CHAPTER 1
INTRODUCTION
FACTORS DETERMINING SUSCEPTIBILITY TO INFECTION
Enviromental and genetic factors play and important role in
determining susceptibility to infection, as well as, its severity. They do so
by interfering in the immunological defence action and the overall
metabolic response.
Age:
Age influences not only susceptibility to infection, but also its clinical
course. Many infectious diseases show a characteristic age distritubion.
Infections with ubiquitous organisms are seen firstly in infants, shortly
after they have lost the maternal antibody transfered across the
placenta, and have a peak incidence in early childhood. Maturation of
the immunological system, associated with gradual acquisition of
protective immunity, due to successive exposures to infection, appear to
promote a more adapted response to infectious threats in adulthood.
With ageing immunity declines increasing susceptibility to infection
again.
Sex and hormonal factors :
Not only hormonal but social factors are implicated in the variation
in the sex distribution of infectious diseases. Sex has a role in defining
occupation and, therefore, can influence how close men and women
come into contact with the source of infection. Pregnancy can
predispose to various infections, including hepatitis, pneumoccocal
infections, amaebiasis and malaria, possibly reflecting changes in
immunological function and in metabolism that occur at that time
(Greenwood , 1983).
The presence of conditions leading to excess glucocorticosteroids,
such as adrenal hyperplasia, adrenal tumors, or steroid therapy, may
increase susceptibility to infections, e.g., infections with herpes simplex
and herpes zoster. Corticosteroids probably exert their action on the
immune and metabolic response ( ). Patients with diabetes, especially if
the lattter in not very well controlled, show increased susceptibility to
infections, with the common development of abscesses, boils and
urinary tract infections. Diabetic patients also present with impaired cellmediated immune reactions and chemotaxis by polymorphonuclear
lymphocytes ( ).
Immunity, Injury and malnutrition :
A number of clinical and experimental studies have shown that
defficiency of protein, of individual vitamins, and of trace elements
increase susceptibility to infection (
). In developing countries, most
attention ought to be paid to children with severe protein-calorie
malnutrition, as these children frequently die from infection ( ). The clear
association of nutrition and infection could recently be seen during the
drought in Ethiopia and other African countries that led to wide-spread
profound malnutrition as the predisposing factor in the death by
infectious disease of tens of thousands of people ( ). In indrustrialized
countries, severe malnutrition is encounted as a complication of
conditions, such as generalized malignant disease, cirrhosis, and
intestinal malabsortion due to inflammatory bowel disease, more
frequently than as a result of pure dietary deficiency. However,
considerable attention has been given to the fact that malnutrition is also
widely prevalent in surgical patients, and in close association with
mobidity and even mortality (Bistrian et al , 1974; Hill et al , 1977 and
1979). Today, careful assessment of the nutritional status, associated
with administration of nutritional support to the malnourished hospitalized
patient is well incorporated into modern clinical care practice (Elwyn,
1980; Kettlewell, 1982; Winters & Greene, 1983; Silk, 1983; Phillips &
Odgers, 1986).
Drugs :
The problem of infection in patients receiving cytotoxic or immuno
supressive drugs is well recognized ( ). Alcohol in excess increases
susceptibility to many infections, especially when liver failure supervenes
( ) . The neglect of simple hygienic precautions when administering their
intravenous injections in hard-line drug addicts leads to septicaemia as a
common terminal event, or alternatively, to the contamination with the
Acquired Immunodeficiency Syndrome virus ( ) . Smoking also
predisposes to respiratory infection due to the damage of the epitheleum
of the respiratory tract ( ) .
Malignant disease :
Immune responsiveness is impaired in patients with dissiminated
malignant disease ( ) . The frequent association of cancer and
malnutrition, added to the harmful effects of radiotherapy or treatment
with cytotoxic drugs further increases the risk of developing infection in
these patients. Whether the provision of nutritional support, with the aim
to correct malnutrition, will have a beneficial effect in diminishing such
risk is still debatable (Dudrick ? ; Copeland, 1986; ).
OBJECTIVE OF THE STUDY
The aim of the work described in this thesis was to study the
important changes in hepatic metabolism induced by sepsis.
Clinical studies of sepsis are difficult to perform, not only due to the
usual restrictions on the actual scope of clinical studies, based on ethical
grounds, but because of the difficulty in establishing comparable groups
of patients. There is usually variability on the severity and type of the
septic insult, which is often associated with variable degrees of trauma,
the latter being commonly accompanied by different nutritional and
hormonal status. Furthermore, it is also not unusual to find variation on
the time the observations are made; and time appears to have a very
important role on the intensity and nature of the changes brought about
by sepsis. Interpretation of the results under these circunstances may,
therefore, be very difficult, for they may reflect, in part, the effects of the
different conditions of experimentation.
In view of the difficulties with patients, we decided to study the time
course of the effects of an experimental model of peritonitis on the
hepatic metabolism of the rat. The particular septic model used has
proved to be very reproducible and clinically sound (Wichterman et al ,
1980). In addition, there appears to be no information on the time course
of the changes in hepatic metabolism in the same experimental model of
sepsis.
The results of the present work may, therefore, help to resolve
some of the apparent discrepancies about hepatic metabolism during
sepsis, found in the review publications in the literature, in both
experimental and clinical sepsis
(Blackburn, 1977; Beisel &
Wannemacher, 1980; Cerra, 1982; Neufeld et al , 1982; Wilmore et al ,
1983; Forse & Kinney, 1985; Frayn, 1986), as illustrated by Fig. 1.1.
Fig. 1.1.
The general term 'sepsis' has caused considerable confusion in
clinical and experimental studies (Wichterman et al , 1980) because it
refers to different conditions in different studies. Some experimental
septic models induce a low-grade smoldering infection which can last for
days or weeks, whereas other models describe a very aggressive
process that produces death whithin hours. For the purpose of the work
described in this thesis, 'sepsis' is regarded as an acute invasive
infection in an animal, which causes the development of a toxic state
(weakness, anorexia, pile erection, lethargia, etc.), leading to the
eventual development of circulatory colapse - septic shock. A wide range
of microbial organisms can cause sepsis, especially gram-positive and
gram-negative bacteria. While the term 'bacteraemia' refers to bacteria in
the blood, irrespective of whether they do or do not induce the toxic state
of sepsis, 'septicaemia' refers to a toxic state produced by an acute
infectious process in the blood. It must be emphasized, however, that
animals dying of infection, with circulatory colapse, with established
septic shock were excluded from the present studies.
THE IMPORTANCE OF INFECTION
Infection was the major factor in worldwide population control for
mankind until only recently. Before the 17th century, epidemics
presumably of smallpox, plague diphtheria, measles, cholera and
infectious diarrheal diseases, were common episodic events causing
death of more than 50% and sometimes as much as 90% within a region
(McNeil, 1977).
Better hygiene and improved nutrition increased the
average life expectancy, and contributed to the five-fold increase in the
world population over the last 150 years (Alexander, 1986). Over this
period, the explosion in scientific methods, associated with the constant
search for controlling and understanding the mechanisms of infection,
have had a tremendous effect on diseases. Smallpox, one of the most
common causes of death in the world a few hundred years ago, is now
extinct (Barnes & Robertson, 1981), whereas e.g. to achieve the control
and eventually the cure for the Acquired Immuno Defficiency Syndrome,
caused by an infection with the human imno-deficiency virus, poses
itselsf as a difficult challenge to the scientific community today (Anon,
1986).
Infection may be part of a primary disease process, for example
pneumonia, or may occur as a complication of surgery, such as a pelvic
abscess following abdominal surgery. However, until the 19th century,
the formation of pus was considered a normal part of healing - pus
bonum et laudbile . John Hunter [1728-1793] recorded in his 'Treatise
on the Blood, Inflamation and Gun Shot Wounds', published in 1794,
that 'inflamation is not only the cause of diseases but it is often the mode
of cure'. Louis Pasteur [1822-1825] caused a dramatic change the
surgical opinion and atitude by showing that fermentation was caused by
living multiplying matter and, therefore, that pus formation and wound
infection had to be also caused by minute organisms from the
enviroment. Joseph Lister [1827-1912] became aware of Pasteur's
research and by the use of carbolic established the principles of
antisepsis. This was a major revolution in surgery which progressed to
the use of aseptic surgical technique and led to a great reduction in
surgical morbidity and mortality. Since Thomas Latta (1832) first
reported in the Lancet
the prevention of death from cholera by the
intravenous injection of 6 pints of almost normal saline containing 3
drachms of salt and 2 scruples of sodium bicarbonate, or about 2.8 litres
of 89 mM Na+, 78 mM Cl-, and 11 mM bicarbonate, the use of fluid and
electrolyte therapy has become such a familiar part of medicine that it is
rarely considered today (Veech R L, 1986). The successiful clinical use
of antibiotics, described for the first time by the Oxford group (Abraham
et al , 1941) was a gigantic step towards the fight against infection.
However, despite the introduction of asepsis and antisepsis, fluid
and
electrolyte
therapy,
specific
antibiotics,
careful
monitoring,
aggressive operative intervention and intravenous nutritional support,
infection still remains a very important clinical problem. Studies have
indicated that about one third of surgical patients suffer from infection at
some stage of their stay in hospital (Altemier et al, 1976; Green et al ,
1977; Cruse & Foord, 1980). Surgical sepsis plays an important role in
the morbidity and mortality encountered in an intensive care unit and
constitutes a major impediment in the resolution of critical ilness, with
septic patients presenting a three-fold mortality rate as compared with
non-septic patients (McLean & Boulanger, 1985). The increasing use of
immunosuppressive drugs and corticosteroids enhances susceptibility to
infection. Despite severely injured patients, transplant patients, patients
with cancer and diabetes melitus are surviving longer now, these
patients have an increased risk of infection. Invasive techniques such as
indwelling intravenous catheters, especially long-term catheters used for
intravenous feeding, and bladder catheters also increase the risk of
sepsis.
The magnitude of the stress imposed by sepsis is a function of
the size of the infectious process, the number of invading organisms and
their virulence. Survival depends not only on the adequacy of the
immunological reactions to contain and eliminate invading microbes, but
also on a series of integrated physiological and metabolic responses
necessary to maintain energy production and cells function thoughout
the body (Clowes et al, 1985). It is encouraging, however, that the
complex pathophysiology of bacterial sepsis is gradually becoming
better understood. The mechanisms of how sepsis becomes a frequent
cause of ventilatory or renal failure , or multiple organ failure, on the
other hand, are yet to be completely clarified.
GENERAL DESCRIPTION OF THE METABOLIC RESPONSE TO
SEPSIS
The course of the metabolic response to trauma, as ilustrated by
Fig. 1.2., was first described by Sir David Curthbertson (1930) more than
50 years ago. These changes will be discussed briefly as they are
similar to those found in the response to sepsis (For review see: Silk,
1983; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986).
Fig. 1.2.
The ebb phase
This phase is characterized by a rapid fuel mobilization,
haemodynamic instability and increased plasma catecolamine
concentrations (Coward et al , 1966). Insulin plasma concentration is
variable (Frayn, 1986) despite a possible inhibition of its release by the
action of the higher catecolamines on the pancreatic ß-cell (Porte &
Robertson, 1973). Glucocorticoids and glucagon are also reported to be
elevated (Forse & Kinney, 1985). Glycogen is mobilised for immediate
energy production but the metabolic rate falls (O'Donnell, 1976).
Although there is some argument about the need, and how adaptive
such a massive fuel mobilization is (Frayn, 1986), maintenance of
volaemia and tissue perfusion appear to be aim during this phase. It is
during the ebb phase, or when conditions similar to those of the ebb
phase are evoked, that septic shock may develop.
The shock of the ebb phase of injury/trauma may be different from
that of sepsis. In the former, there is decreased energy production
associated with hypovolaemia, whereas in the ebb phase of sepsis, the
'septic shock' may present in a hyperdynamic state, with increased
energy production, hyperventilation, increased central venous pressure,
increased cardiac output, oliguria, decreased peripheral resistance,
hypotension and lactic acidosis (Siegel et al , 1967; Forse & Kinney,
1985). This hyperdynamic septic state then evolves to the hypodynamic
septic state with low central venous pressure, hypotention, low cardiac
output and increased peripheral resistance (MacLean, 1981; Forse &
Kinney, 1985). This suppports the view of a bimodal response to severe
acute sepsis, with an initial hypermetabolic hyperdynamic period
followed by a hypometabolic hypodynamic late septic state (Chaudry et
al , 1979; Wichterman et al , 1980).
The flow phase
If death does not occur as a result of hypovolaemia or direct
damage to vital organs during the ebb phase, a more prolonged period
follows (the flow phase), which is characterized by an increase in the
metabolic rate, breakdown of body tissue and weight loss. Increased
plasma concentrations of insulin and glucagon have been reported
during this phase (Biesel & Wannemacher, 1980; Neufeld et al , 1982).
Generally this phase of the metabolic response can be regarded as a
balance between the action of insulin and those of the counterregulatory
hormones, namely, glucocorticoids, glucagon and catecholamines
(Forse & Kinney, 1985). Another characteristic feature of this phase is
the presence of hypermetabolism (Cuthbertson, 1979), which appears to
be proportional to the degree of stress (Elwyn et al , 1981) or the
severity of infection. So major infections such as peritonitis are
associated with increases of up to 60% above the resting energy
expenditure (Elwyn, 1980).
The aim of this hypermetabolic catabolic
phase appears to be to maintain energy production and cell function
thoughout the body, at a time dietary intake may be scarce and/or not
absorbable. If this is acheived and the immunological and metabolic
responses take a successiful course, the changes leading to net
catabolism are gradually replaced by those leading to net anabolism,
merging into the 'anabolic' or 'convalescent' phase.
The anabolic phase
The transition from the catabolic flow phase to the anabolic period
of convalesce can occur quickly, within a few days, as with soft tissue
injury or can occur over several days as with extensive trauma (Moore &
Brennan, 1975). The distinct metabolic changes that occur during
anabolism aim to replace the tissue lost over the catabolic phase,
whereas resting energy expenditure may fall from the elevated levels of
the flow phase to subnormal levels, similar to those found in depleted
patients (Moore & Brennan, 1975; Forse & Kinney, 1985).
However, while the pattern of the response to injury shows a
defined time-sequence, from the trauma itself until recovery, the course
of the response to sepsis is not so predictable, because of fluctuations in
the nature and intensity of the metabolic changes, which are linked with
the severity, the type of infection, and variation in the nutritional and
hormonal state. Take, e.g., a patient with intra-abdominal sepsis
proceeding well during the flow phase. If part of the avascular fibrinous
wall of a previously isolated-off abscess is
eroded bacteraemia can
develop, which in turn, will precipitate events of the ebb phase leading or
not to the establishment of a 'septic shock' state.
SEPSIS AND ENDOTOXAEMIA
Endotoxin is a lipoprotein-carbohydrate complex (LPS) found in the
cell wall of all gram-negative bacteria. Andre Boivin (1941) was the first
to isolate endotoxins from gram-negative bacteria, and some ten years
latter endotoxins were implicated as the cause of human gram-negative
shock (Borden & Hall, 1951; Braude, 1953). This hypothesis has led to
numerous studies on the physiological and biochemical alterations
associated with endotoxin inocculation into various laboratory animals
over the last three decades. Furthermore, some of the results of such
studies
are
usually
incorporated
into
the
descriptions
of
the
pathophysiology of clinical sepsis and septic shock, and in their
treatement (Wichterman et al , 1980).
Some of the clinical aspects that result from gram-negative sepsis
and endotoxin desagree (Fig. 1.3.), as described by Wichterman et al
(1980). Nevertheless, both endotoxin and sepsis can activate the
macrophages to produce interleukin-1 and/or its by products, which
appear to be important mediators of the metabolic changes brought
about by sepsis and/or endotoxin (Fig. 1.4. - Cohn, 1978; Clowes et al ,
1983), and can cause similar hormonal and metabolic changes, such as,
hyperinsulinaemia and hyperglucagonaemia (Yelich & Filkins, 1980;
Beisel
&
Wannemacher,
1980),
and
the
inhibition
of
the
hyperketonaemia in response to starvation (Neufeld et al , 1982). The
development of multiple organ failure syndrome also appears to follow
both endotoxaemia (Tracey et al , 1986) and sepsis (Duff, 1985).
Therefore,
data
concerning
endotoxaemia,
relevant, will be included in this manuscript.
Fig. 1.3.
whenever
considered
On one hand, there appears to be no evidence that endotoxin has a
direct active role in human shock (Baue et al , 1969). In addition, very
few septic patients have endotoxin identified in their blood, and if so, at
concentrations which are quite low (Levin et al , 1972; Postel et al ,
1975). On the other hand, recent studies support the view endotoxin acts
in an indirect way via stimulation of polymorphonuclear leucocytes
(Tracey et al , 1986; Beutler & Cerami, 1987; Christou et al , 1987),
which may help to explain some of the differences in the clinical findings.
MULTIPLE ORGAN FAILURE
The failure of vital organs in the septic and/or injured patient has
received the attention of a number of recent studies (for review see:
Polk, 1982; Duff, 1985). It is clear now that multiple organs are often
involved, usually remote from the site of infection. In addition, the clinical
manifestations of their involvement show a sequential pattern. Failure of
the renal, respiratory and cardiovascular systems often occur first, and, if
this is not followed by recovery, there is failure of the brain, liver and gut
(Duff, 1985).
The lungs
The earliest recognized form of organ failure was that involving the
lung. Adult respiratory distress syndrome (ARDS), 'shock lung', 'Viet
Nam lung' and 'lung lesion of sepsis' all describe a type of respiratory
failure, with each name being related with their initiating event: shock,
trauma and/or sepsis (Polk, 1982). The predominat characteristic of
adult respiratory distress syndrome appears to be the associataion with
infection ( Horovitz & Shires, 1974; Eiseman & Narton, 1975; Fulton &
Jones, 1975). Fulton & Jones (1975) showed that 85% of their patients
had clinically identifiable sepsis, before or along with the establishment
of pulmonary dysfunction. The pathophysiology of lung failure is not
clear, but there is some evidence from clinical and experimental studies
that the development of the pulmonary oedema is a result of increased
permeability of pulmonary capilaries. Increased capillary parmeability
has been reported in sheep injected with endotoxin or bacteria, as
judged by the increase in the rate of appearance and the concentration
of protein in the lung limph (Brigham et al , 1979). Adult patients with
ARDS showed increased rate of incorporation of blood radiolabelled
albumin into the broncheoalveolar fluid (Sibbald et al, 1979). The
mechanism of cappilary injury is not fully explained, but the effects of
complement activation may play an important role (Creddock et al, 1977;
Jacob et al , 1980). This endothelial cell injury could also be the result of
the action of the polymorphonuclear leucocytes (or their products) which
are konwn to be aggregated to activated complement. To support this
view there is evidence that drug-induced neutropoenia can diminish or
prevent the effects of infusion of activated complement (Craccock et al,
1977). Other causative factors are also likely to be involved, as
experimental lung lesions caused by infusion of complement are never
as extensive as those observed in septic patients (Hyers, 1981). Using a
radioaerosol lung scanning technique, Tennemberg et al (1987) studied
patients with trama and sepsis, and reached the conclusion that
complement-mediated neutrophil activation alone appeared to be unable
of initiating lung injury or precipitating ARDS. Complement-mediated
neutrophil activation may, therefore, serve as an early, potentiating
mechanism for other polymorphonuclear triggering mediators. It is also
well known that the lungs, in addition to their gas exchange and filter
function, and the fact they are target-organs of circulatory factors, they
clear many substances from the circulation such as 5hydroxytryptamine. The lungs also synthesise and release various
agents, that may act on smooth muscle and, therefore, interfere with the
function of other organs; the lungs being thus regarded in a way
analogous to an "endocrine gland" (Aun & Birolini, 1985).
The kidneys
The pathophysiology of the injuries leading to renal failure is not
fully understood. Acute tubular necrosis may or may not be present.
Decreased renal blood flow, endotoxin action and vasocontriction of
renal arteries have been implicated as possible mechanisms (Duff,
1985). Fortunately renal failure can be treated with success with
haemodialysis until recovery of function returns, but the latter is usually
linked to the recovery of function of other organs, especially the liver.
The liver
Severe sepsis causes liver damage and liver function is impaired in
the multiorgan failure syndrome. Liver function tests show a pattern
suggestive of cholestasis with a rise in serum bilirubin and alkaline
phosphatase, accompanied by moderate elevation of SGPO and SGPT
(Royle et al , 1978). Jaundice is the clinical sign which reflects such
changes, and appears at a later stage, as compared to the biochemical
changes. Changes in blood amino acid concentrations include
elevations of phenylalanine, taurine, cystine and methionine, which are
metabolized in the liver (Freund et al , 1978). Macroscopic changes in
livers of monkeys, injected with live E. coli bacteria, include intravascular
sequestration of degranulating leucocytes and fibrinous deposits of
platelet agregates in synusoids and the spaces of Disse (Balis et al ,
1979). The post-morten of patients who died with multi-organ failure
reveals hapatic changes such as, focal necrosis, fatty accumulation ,
congestion and enlargement of synusoids, and cholestasis (Champion,
1976).
Gastrointestinal system
The gastrointestinal (G.I.) system may fail due to ulceration, loss of
barrier function or malabsorption. In patients dying from multi-organ
failure, the post-morten shows massive denudation of the
gastrointestinal tract mucosa, which suggests a failure in the
gastrointestinal barrirer function, enhancing abnormal absorption of
bacteria and/or bacterial products (toxins) (Duff, 1978).
Pathophysiological mechanisms of multiorgan failure syndrome
A common mechanism used to explain the development of
multiorgan failure is found on the action of free radical (O2-, OH-,
H2O2). As a result of sepsis, or sepsis associated with trauma (usually
represented by the operation to treat localized sepsis), there is activation
of the complement cascade through the alternative pathway, and release
of factor C5a (a polypeptide of 74 residues and a molecular weight of
15,000). This peptide would interact with circulating neutrophils to
increase their adherence and probably to degranulate when exposed to
suitable stimuli (Simmons & Solomkin, 1985). The agregation of
granulocytes would, in turn, release free radicals in ammounts sufficient
to cause cellular damage, dysfunction and eventualy organ failure (Duff,
1985).
Recent evidence, however, appears to indicate a very important
role of the macrophage in the response to sepsis. Activated
macrophages by bacteria or bacterial products, e.g. endotoxin
[lipopolysaccharide
(LPS)],
can
release
a
polypeptide
hormone
(cachetin) which might initiate tissue injuries, the latter being not different
from those caused by endotoxin inoculation (Tracey et al , 1986; Beutler
et al, 1987).
THE ROLE OF PHAGOCYTIC CELLS
Phagocytic
cells,
including
neutrophils,
monocytes
and
macrophages, are responsible for the process of phagocytosis. At the
begining of this century Metchnidoff (1905) described phagocytosis,
namely as the ingestion and subsequent killing of micro-organisms
(endocytosis), and believed this was a very important process in both the
prevention and fight against infection. Latter on, however, it became
clear that an important contribution of phagocytes to the inflamatory
response is the exocytosis of enzymes and other proteins, some of them
possibly carriers of important signals for the development of this
response (inflammatory or humoral mediators). Exocytosis, on the other
hand, can occur independetly from endocytosis, in response to different
stimuli from those of the latter (Bentwood & Henson, 1980).
These proteins released by the inflammatory cells are capable of
various actions, such as making accessible the site of inflammation from
the adjunct vascular space (e.g. collagenase and elastase); regulating
local inflammatory mediators (e.g. carboxypeptidase); controlling the
concentration of metabolites involved in bacterial growth (e.g. lactoferrin
and vitamin B12); and enhancing an extracelluar environment adverse to
micro-organisms through the action of acid hydrolases, cationic proteins
and lysozymes (Simmons & Solomkin, 1985).
THE MACROPHAGE
Macrophages are distributed throughout the body and have
developed specific characteristics in various organs, from which they
acquired their respective names (Kuppfer's cells within the liver,alveolar
macrophages in the lung, messengial cells in the kidney, and glial cells
(which are thought to be derived from macrophages) in the brain. It
appears to be clear that macrophages have adapted for local defence
purposes, and in addition, macrophage function can also vary at different
sites (For reviwe sse: Takemura & Werb, 1984). The three primary
functions of macrophaghes, as described by Cristou et al (1987), are
listed bellow:
1. Immune function
Antigen presentation
2. Phagocytic-microbicidal function
Phagocytosis
Destruction of viral infected cells
Oxidative killing mechanisms
Chemotactic factors
Lysozyme relase
Destruction of intracellular parasites
3. Secretory function
Proteases
Procoagulant activity
Interleukin 1 (IL-1)
Colony stimulating factor
Angiogenesis factor
Plasminogen activating factor
Prosaglandins/leukotrienes
Interferon
Tumor necrosis factor (TNF)/cachetin
"Hepatocyte modulating factor(s)"
The differences in macrophage function could also be understood
in terms of degree of macrophage activation, namely, how 'stimulated'
the macrophage is. A spectrum of stimulation can be recognized
depending on the potency of the stimulating agent. Thus, macrophages
which are stimulated with e.g. interferon or complement are reffered to
as "inflammatory" or 'elicited" macrophages, whereas macrophages
stimulated, for example, by endotoxin (Lipopolysaccharide - LPS) are
fully "activated" macrophages (Cohn, 1978). The activation of
macrophages results in enhanced phagocytosis (Cohn, 1978), increased
production of toxic oxygen radicals (Tsunawaki et al , 1974) and the
release of various proteases and hydrolytic enzymes (Takomura &
Werb, 1984). There is also release (Fig. 1.4.) of various monokines
(Takamura & Werb, 1984; Dinarello et al, 1986; Aggarwal et al , 1985;
Beutler et al , 1986; Kovaks et al , 1985; ).
Fig. 1.4.
Release of oxygen radicals by activated macrophages (Babion et
al, 1973; Craddock et al, 1977) has also been reported in sepsis.
Interleukin 1 can be produced by virtually all activated macrophages
(Dinarello & Mier, 1986) and has been implicated as an important
mediator in the hypercatabolism of proteins in response to sepsis
(Baraco et al, 1983; Clowes et al, 1983 and 1985). Macrophages treated
with endotoxin (LPS) in vitro show altered secretion of various proteins
(Largen et al, 1986). An important monokine released by endotoxin
stimulation of macrophages is the Tumor Necrosis Factor (cachectin),
which appears to be an important humoral mediator of the systemic
metabolic changes during sepsis (Butler et al, 1985; Cerami et al, 1986).
Studies using co-culture of hepatocytes with Kupffer's cells or
macrophages, in the presence or absence of inflammatory stimulation,
showed that there was a significant depression of hepatocytes protein
synthesis when endotoxin (LPS) was added to the co-culture medium.
However, in the abscence of either macrophages or Kupffer's cells,
endotoxin had no direct effect on hepatocytes protein synthesis (West et
al, 1985 and 1985). More recent evidence suggests the macrophagemediated inhibition of protein synthesis in isolated liver cells to occur via
a monokine mediator yet to be identified, but not Interleukin 1 or
cachectin (Christou et al ,1987).
The importance of macrophages in response to sepsis can also be
demonstrated by the important role of resident macrophages in the
peritoneal cavity to fight bacteria and avoid peritonitis. Within 20 min of
bacterial injection into the peritonial cavity, 35 to 45% of the bacteria
inoculated will be associated with macrophages (Dunn et al, 1987).
Baker and Kupper (1985) using the caecal-ligation and puncture (19gauge needle) model in two strains of mice, showed 100% mortality in
septic endotoxin-sensitive mice (C3H/HeN), whereas the moratlity of
septic endotoxin-insensitive mice (C3H/HeJ) was only 25%, within the
same period of time after the induction of sepsis (4 days). These two
strains of mice differ only in one gene, which controls some aspects of
macrophage function (Adams et al, 1981). Therefore, although
macrophages are necessary for local protection against sepsis,
infection-stimulated macrophage secretory products may also be
responsible for the induction of some of the irreversible changes of the
response to sepsis, e.g. multiple organ failure.
THE ROLE OF INTERLEUKIN 1
Interleukin 1 is critically important in the normal immune response
by allowing T-helper cells to differentiate and secrete Interleukin 2, which
is required for numerous lymphocyte effector functions (Fig. 1.4.).
Interleukin 1, or its by-products, have been suggested as the link
between the immunological and the metabolic response to sepsis
(Clowes et al, 1986). Interleukin 1 is a polypeptide secreted by
macrophages (molecular weight 14,000 - for review see: Dinarello &
Wolff, 1982; Dinarello & Mier, 1986). Because the biochemical nature of
every macrophage-derived factor is not yet well defined, some of these
factors, initially described by their performance in different functions,
may actually be the same molecule. There is some evidence now
Interleukin 1 plays multiple roles (Takemura & Werb, 1984). It can act as
a potent chemotaxin for neutrophils and enhance endothelial
adhesiveness. It has also effects on fibroblast proliferation and may be
used in the wound healing (Dinarello, 1984). Endogenous pyrogen,
which acts on the hypotalamus to change the set point of body
temperature (Baracos et al, 1983) and acute phase protein syntesis
promoting factor (Stein et al, 1981) seem to be Interleukin 1. Muscle
proteolysis induced by sepsis appears to be stimulated by a circulating
clevage product of Interleukin-1, namely, proteolysis inducing factor
(Clowes et al, 1983 and 1985). It has been suggested leucocytic
pyrogen (interleukin-1) or proteolysis-inducing factor may act in the
muscle by increasing the production of prostaglanding E2 (PGE2) which,
in turn, would activate lysosomal proteases by a calcium depending
mechanism (Rodeman et al , 1982; Freund et al, 1986). PGE2 is
derived from arachinodate via the cyclooxygenase reaction (Rodeman et
al , 1983). Therefore, cyclooxygenase inhibitiors, such as indomethacin
and aspirin, might be useful in atenuating or decreasing muscle
proteolysis induced by sepsis.
Haemodynamic abnormalities and survival rates have been
reported to be improved by the use of indomethacin in various endotoxin
and septic models (Fletcher et al , 1976 and 1977; Short et al , 1981;
Fletcher, 1982 and 1983).
However, there is evidence that
administration of indomethacin to rats, subjected to caecal-ligation and
puncture, did not affect the increased muscle proteolytic rate found in
these septic rats. When indomethacin was added in vitro release of
PGE2 by septic and normal muscles, incubated in the presence of septic
plasma, was reduced by 50%, but the increased proteolytic rate in these
muscles remained unchanged (Hasselgren et al , 1985). On the other
hand, Freund et al (1986) showed that whole body protein degradation
was significantly lower in indomethacin-treated septic rats, but this was
associated with lower plasma albumin and decreased protein synthesis
in muscle and liver, at a time new protein production is essential,
therefore not supporting the use of indomethacin in sepsis. Miccolo et al
(1986) using pharmacological dosage of aspirin, given to septic patients,
and measuring urea production rate through the incorporation of 15N
into the plasma pool, showed no effect of aspirin on the protein
breakdown rate in septic man.
Crude
leucocytic
endogenous
mediator
(LEM)
consists
of
substances derived from polymorphonuclear leukocytes, which include
endogenous pyrogen (interleukin-1), and is reponsible for inducing
acute-phase reactant production in the liver and changing the plasma
concentrations of certain divalent cations, namely, iron and zinc
downwards and copper upwards (Dinarello, 1984; Kampschmidit, 1984).
LEM is also able to induce hyperinsulinaemia, hyperglucagonaemia,
glycogen depression, and other metabolic changes similar to those
found in sepsis (George et al , 1977).
Interleukin-1, in vitro , did not affect the inhibited muscle amino acid
uptake in response to sepsis (Hassselgren et al, 1986). However,
interleukin-1 injected into the rat in vivo , had a stimulatory effect on
hepatic metabolism, in isolated liver cells, in a time-dependent pattern
(Roh
et
al,
1986).
These
changes
included
stimulation
of
gluconeogenesis from alanine[0.5mM], elevation in non-secretory protein
synthesis and increase in oxygen consumption. If interleukin-1 was
added to the hepatocyte incubation medium, however, no stimulatory
effect was obtained, suggesting the effects of interleukin-1 upon isolated
hepatocytes are indirect. This view of indirect action of interleukin-1 in
the liver, in vitro, is further supported by the fact that interleukin-1, when
added to hepatocytes' culture alone, or to hepatocytes co-cultured with
Kuppfer's cells, had no effect on rates of protein synthesis (Christou
et al ,1987).
CACHECTIN
Cachetin (or Tumor Necrosis Factor)
is a polypeptide hormone
secreted by the macrophage, originally isolated in the course of studies
of the mechanisms reponsible for cachexia in chronic disease, and
known for its capacity to modulate adipocyte metabolism, to lyse tumor
cells in vitro , and to induce hemorrhagic necrosis of certain
transplantable tumors in vivo (for review see: Tracey et al , 1986; Beutler
& Cerami, 1987). It has a sub-unit size of approximately 17 kilodaltons
and constitutes between 1 and 2 % of the total secretory protein
produced by endotoxin-activated macrophages in vitro (Beutler et al ,
1985).
The role of cachetin as a mediator of endotoxic shock was
suggested by the fact mice passively immunized against the hormone
were found to be protected against the lethal effect of endotoxin
(lipopolysaccharide), as reported by Beutler et al (1985). Tracey et al
(1986) infused cachectin into rats, in quantities similar to those produced
endogenously in response to endotoxin, causing piloerection and
diarrhea. Haemoconcentration, shock, metabolic acidosis, transient
hyperglycaemia followed by hypoglycaemia, and hyperkalaemia were
also observed. Gross and microscopic examination demonstrated
severe damage to target organs. Adrenal and pancreatic hemorrhages
were commonly observed. Polymorphonuclear leukocytes thrombi were
deposited into the major arteries of the lungs, and this was associated
with severe pneumonitis. Administration of low doses of cachectin (100
to 200 µg/kg body wt. in rats) induces renal tubular necrosis, and
ischemic and hemorrhagic lesions in the gastreointestinal tract (Beutler
& Cerami, 1987).
To minimize inadvertent release of cachetin, its synthesis appears
to be tightly regulated as both transcriptional and post-transcriptional
activation must occur to allow its production (Beutler et al , 1986).
Glucocorticoids strongly antagonize the effects of endotoxin when
administered before the endotoxic insult. If glucocorticoids are added to
macrophage cultures cachectin synthesis will be completed inhibited.
However, glucocorticoids will only promote this action if added to the
culture before macrophages are activated by endotoxin. Therefore, no
inhibiting effect occurs if dexametasone is added after lipossacaride
induced-macrophage activation.
It has been shown cachectin appears to act by suppressing
biosynthesis of several adipocyte-specific mRNA molecules (Torti et al
,1985). Cachectin can also induce the synthesis and/or release of
specific proteins, including interleukin-1 by monocytes and endothelial
cells (Dinarello et al , 1986; Nawroth et al , 1986). Cachectin can activate
polymorphonuclear leukocytes stimulating their adhesion to endothelialcell surfaces and enhancing their phagocytic activity (Gamble et al ,
1985; Shalaby et al , 1985). Some other effects that may explain the
action of cachectin in vivo include stimulation of the production of
procoagulant activity altering the properties of the vascular endothelium
and alteration of patterns of antigenic expresion (Beutler & Cerami,
1987).
Specific neutralization of cachectin and related cytokines may offer
new therapeutic tools to treat sepsis. It has been shown that mice
treated with a polyclonal antiserum directed against mouse cachectin
become resistant to the lethal effect of endotoxin (Beutler & Cerami,
1985). Therefore, it would seem possible that neutralizing monoclonal
antibodies directed against human cachectin may prove to be useful in
the treatment of sepsis, particularly in its early stages.
THE ROLE OF STEROIDS
The complex balance between the anti-inflammatory properties and
the physiological requirement for steroids may explain the conflicting
conclusions about steroid therapy in sepsis (Hess & Manson, 1983; Nohr
& Meakins, 1985). Although controversial, the clinical use of steroids in
septic shock is common practice and large doses of steroids (50-150
mg/kg body wt.) have been given (Forse and Kinney, 1985). The
rationale for the use of these agents in sepsis has included the ability of
these drugs to stabilize lysosomal and cell membranes, to inhibit
complement-induced granulocyte aggregation, to improve myocardial
performance
(possibly
due
to
inhibition
of
oxygen-free
radical
generation), to modulate the release of endogenous opiates, to decrease
arachidonate metabolies, and to promote a shift in the oxyheomoglobin
dissociation curve to the right (Karakusis, 1986).
Corticosteroids are important in maintaining normal physiology and
their concentration may or may not be increased in response to sepsis
(Sibbald et al , 1977). Elevation in steroid levels during sepsis may be a
protective response to decrease tissue damage by modulating
inflammation. The effects of steroids on specific immune processes are
also complex. Leucocyte traffic is altered and specific antibody
responses are either not affected or enhanced by steroids (Nohr &
Meakins, 1985). In the liver, pharmacological doses of steroids have
been shown to diminish the disruptive effects of gram-negative
bacteraemia and endotoxin on the reticuloendothelial system (Balis et al
, 1979).
Steroids inhibit margination and degranulation of
polymorphonuclear leucocytes in the microcirculation of the liver, lung
and other organs, and may
therefore be able to inhibit complement
activation and the release of factors which decrease capillary
permeablity, the latter being implicated with the pathophysiology of
multiple organ failure syndrome (Duff, 1985).
Steroids block the synthesis of prostaglandins by inhibiting the
release of arachidonic acid from membrane phospholipids, and this may
be one mechanism by which steroids exert their beneficial effect in
septic and shock (Carlson et al , 1977; Fletcher, 1982; Halla-Angeras et
al , 1986). As arachidonate is the substrate of both the cyclooxygenase
and the lipoxygenase pathways, steroids also may inhibit the synthesis
of leukotrienes (Samuelsson, 1983). Recent evidence has shown a
protective action of dietylcarbamazine, an inhibitor of leukotriene
biosynthesis, against the lethal effect of endotoxin in mice (Hagmann et
al , 1984), given support to the view of leukotriene inhibition as another
possible alternative mechanism steroids may use to promote their
beneficial actions during endotoxin and/or infection. Hall-Angeras et al
(1986) have shown that survival rate of rats, subjected to laparotomy
and intravenous infusion of live E. coli, was significantly improved by
methylpredinisolone or diethylcarbamazine. However, this protective
effect appeared not be related to inhibited prostaglandin synthesis, but
might have been due to inhibited production of leukotrienes.
Glucocorticoids also act as important positive modulators of hepatic
and renal gluconeogenic enzymes, as they play a permissive role in the
induction of enzymes by other hormones, such as glucagon and
epinephrine (Shackleford et al , 1986). There is evidence that sepsis or
endotoxin treatment are able to inhibit gluconeogenesis (Filkins &
Cornell, 1974; Guillem et al , 1982; Clemens et al , 1984). Investigations
into the cause of impaired gluconeogenesis have shown a blockage in
the glucocorticoid induction of important gluconeogenic enzymes,
including
glucose-6-phosphatase,
tryptophan
oxygenase
and
phosphoenolpyruvate carboxykinase (Ripe and Berry, 1972; Guckian,
1973; Cannonico et al , 1977; Shackleford et al , 1986). Therefore,
interest has been growing on the mechanisms of the endotoxinglucocorticoid antagonism. Moore et al (1976, 1985) have determined
that inhibition is elicited by a protein mediator released from
macrophages,
named
"glucocorticoid-antagonizing
factor"
.
Glucocorticoids are known to act via intracellular receptors to which they
bind with high affinity after diffusion through the cell membrane (Higgins
& Gehring, 1978).
These steroid-receptor complexes suffer
conformational change ("activation"), and then bind to acceptor sites in
the nucleus, resulting in enhanced transcription of a small percentage of
genes (Schmidt & Litwack, 1982). Shackleford et al (1986), studying
the hormonal regulation of phosphoenolpyruvate carboxykinase in
endotoxin treated mice, have demonstrated no effect of endotoxaemia
on binding, and on affinity or number of glucocorticoid receptor binding
sites, rulling out down-regulation of receptors as the mechanism
responsible for the inhibition of induction. They reached the conclusion
that endotoxin does not act at the early events in the glucocorticoid
induction process, but might act at a subsequent step.
Very recent evidence shows glucocorticoids are able to completely
inhibit cachetin, a hormone released by endotoxin-activated
macrophages, provided they are applied to macrophage cultures in
advance of activation (Beutler & Cerami, 1987).
THE COUNTER-REGULATORY HORMONES
There are difficulties in understanding hormonal mechanisms
involved in producing the responses of flow phase of injury / sepsis, and
in fitting the time-course of the action of a specific hormone, or group of
hormones, to the time-sequence of the metabolic changes (Frayn,
1986).
Because of their known general catabolic effects, the counterregulatory hormones, including adrenaline, cortisol, glucagon and growth
hormone (Cryer, 1981), have received considerable attention in the
study of sepsis. The action of these hormone, in concert, can produce
stimulation of lipolysis, glycogenolysis and gluconeogenesis (Frayn,
1986). Infusion of catecolamines associated with glucagon and cortisol
has been shown to mimic most of the metabolic features of the flow
phase after injury, such as increased metabolic rate with elevation of
glucose turnover, increased gluconeogenesis and negative nitrogen
balance (Bessey et al , 1984; Gelfand et al , 1984). However, contrary to
the view of the counter-regulatory hormones being the main regulators of
the changes of the flow phase, is the fact that neither the concentrations,
or the time course of changes in the concentrations of these hormones
in injured patients agree with those described in the infusion studies
(Frayn, 1986). Watters et al (1986) infusing hydrocortisone, glucagon
and epinephrine, associated or not with the injection of an inflammatory
agent (etiocholanolone), to healthy volunteers reached to the conclusion
that both inflarmmatory and endocrine mediators are necessary to
stimulate the responses to sepsis.
SEPSIS AND INSULIN SECRETION
Insulin is perhaps the most studied, but the least understood
homone in its role during the acute response to sepsis. Many of the early
features of the response characterized by fuel mobilization, such as,
complete use of glycogen reserves and mobilization of triacylglycerol
might be explained by a decrease of insulin secretion (Frayn, 1986),
possibly due to supression of insulin release through the effect of higher
concentrations
of
catecolamines,
found
in
sepsis
(Biesel
&
Wannemacher, 1980), on the pancreatic ß-cell (Porte & Robertson,
1973). However, although plasma insulin concentration has been
reported to be decreased in experimental sepsis (Kelleher et al , 1982;
Lang et al , 1984) and in the septic man (Clowes et al , 1966), it has also
been shown to be increased during experimental sepsis (Neufeld et al,
1980 and 1982; Biesel & Wannemacher, 1980) and in the septic man
(Long et al ,1985; Shaw et al, 1985). Higher concentrations of insulin
have also been reported during endotoxaemia (Yelich & Filkins, 1980;
Neufeld et al , 1982). This variability in insulin concentration may be
explained by differences in the experimental model of sepsis used, the
changes in the type and source of sepsis, nutritional status and the timepoint observations were made. Despite this variability, the metabolic
responses seem to be relatively uniform and insulin, therefore, appears
to act ineffectively during infection being unable to produce its anabolic
effects.
Insulin Resistance
One unified way of looking at the endocrine control of the
metabolic changes after injury/sepsis is the presence of an
unresponsiveness of the 'net storage' or the anabolic processes to the
elevation in insulin concentration. Therefore, glucose production in vivo
appears to be enhanced while glucose storage is reduced in the
presence of high insulin (Black et al , 1982). Despite insulin to promote
normal fat storage, net fat mobilization and oxidation are increased
(Stoner, 1983; Frayn et al , 1984). To further ilustrate the failure of
proper insulin action is the finding of a striking positive relationship
between the time course of the increase in plasma insulin with the
amount of nitrogen excretion, in injured patients (Frayn, 1986). Despite
the general concept of insulin resistance during injury/sepsis has been
supported for many years (Butterfield, 1955; Hinton et al , 1971; Beisel &
Wannemacher, 1980) there is a lack of understanding of the insulin
action at the tissue level, from receptor through possible intermediates to
target pathways. Black et al (1982) using a modification of the
euglycaemic insulin clamp technique to study injured patients, suggested
that the sensitivity of the insulin receptor to circulating insulin is normal,
and that insulin resistance in peripheral tissues (probably skeletal
muscle) may be caused by a postreceptor defect. Clemmens et al (1984)
studying isolated perfused livers showed a resistance to the effects of
insulin on gluconeogenesis in the livers of septic rats (caecal-ligated and
punctured rats), as compared to those found in the livers of shamoperated rats. Hasselgren et al (1987) reported some evidence for
insulin resistance of protein breakdown in septic muscle, while the
response to the hormone of amino acid transport and protein synthesis
was not altered in sepsis.
BIOLOGICAL RESPONSE MODIFIERS
All treatments, procedures and agents used to restore
homaeostasis and maintain normal anatomy and physiology are
regarded as biological response modifiers (For review see: Meakins,
1981; Nohr & Meakins, 1985). Despite the difficulties in selecting the
appropriate modifier, as well as, in evaluating their action, they appear to
hold much promise in the modulation of the metabolic response to
injury/sepsis. A list of some of the biological response modifiers is shown
below (Nohr & Meakins, 1985).
Biological agents
Endogenous
Tissues and cells
Hormones
Passive immunization
Target-directed monoclonal antibodies
Miscellaneous
Exogenous
Specific: active immunization
Non-specific: adjuvants
Drugs
Corticosteroids
Free radical sacvengers
Iron binding compounds
Prostaglanding related drugs
Levamisole
Opiate antagonists
Insulin release inhibitors
Others
General Methods
Restore anatomy and physiology
Surgery
Nutrition
Plasmapheresis
Fever
Some of these modifiers will be briefly considered. Thymic extracts,
namely a derivative of thymopoetin, TP5, increases survival in a septic
burned animal model (Waymak et al , 1984).
The role of some hormones (steroids and counterregulatory
hormones) has already been mentioned in this introductory chapter.
The important role of endotoxin (LPS) in the pathogenesis of sepsis
suggests that antibody to LPS may be therapeutically useful as an antitoxin. However, the most immunologically accessible poetion of LPS (the
O
antigen
side
chain)
varies
among
gram-negative
bacteria.
Nevertheless, animal models and a human trial using human anti-E coli
J5 antiserum have shown protection against the lethal effects of gramnegative sepsis (Dunn & Ferguson, 1982; Ziegler et al , 1982).
It has also been demonstrated (Beutler et al , 1985) that mice
treated with a polyclonal antiserum directed against mouse cachectin
become resistant to the lethal effect of lipopolysaccahride (LPS). Thus
the use of monoclonal antibodies may also have potential therapeutic
applicability.
Fibronectin is an opsonic glycoprotein important in promoting
phagocytosis by cells of the reticuloendothelial system. Plasma levels of
this glycoproetin are decreased in septic patients. Scovill et al (1978)
have shown that the administration of fibronectin as cryoprecipitate can
restore some aspects of host physiology towards normal.
Zymosan, a cell wall preparation of Saccharomyces cerevisiae
which can attract and activate leucocytes enhancing their phagocytic
properties (Williams et al , 1982), has been used as a pre-treatment
(injected intraperitoneally) to rodents and successifully decreased
mortality due to peritonitis (Joyce et al ,1978). On the other hand, Goris
et al (1986) advocate the use of zymosan, also intraperitoneally, to
induce multiple organ failure and 'sepsis without bacteria'.
The role of prostaglanding inhibitors in the modulation of the
metabolic response to sepsis has also being discussed previously.
Opiate receptors in the central nervous system may mediate some
effects of septic shock, possibly in conjunction with opiate receptors in
the periphery (Nohr & Meakins, 1985). Administration of the opiate
antagonist, naloxone hydrochloride, can alleviate the hypotension of
endotoxin shock to varying degrees, to increase mean and pulse blood
pressure,
elevate
respiratory
rate,
prevent
acidosis,
attenuate
hypoglycaemia, stabilize lysosomal membranes and elevate white blood
cell and platelet concentrations (Groeger et al , 1983; Hinshaw et al ,
1984; Ipp et al , 1984). However, some of the reasons to question their
value as a therapeutic intervention for septic shock include the fact that
studies evaluating survival time have shown no long-term survival
benefits; naloxone cardiovascular's effects have been mostly assessed
only during the early phase of shock; the effects on cardiovascular
parameters due to naloxone are often short-lived and minimally
beneficial; despite increasing arterial pressure naloxan may also present
adverse actions, such as regional vascular resistance; species variability
has been reported with regard to some of naloxone's vascular actions;
and finally, naloxone stimulates the sympathoadrenal system which may
impair perfusion of the microcirculation (Hinshaw et al , 1984).
Hyperinsulinaemia is a common event as part or the metabolic
response to sepsis (Biesel & Wannemacher, 1980). One unified view of
the metabolic changes during sepsis is the lack of response to the
anabolic action of insulin (Frayn , 1986). Hyperinsulinaemia, itself, has
been implicated as the cause, rather than the product of insulin
resistance (Marangou et al , 1986). Therefore, drugs modulating insulin
release can also be used not only to assess a possible reversal in insulin
sensitivity during sepsis, but also to try to reverse some of the metabolic
changes mediated under the conditions of elevated plasma insulin.
Diazoxide (Henquin et al , 1982), mannoheptulose (Simon et al , 1972)
and streptozotocin (Bolaffi et al , 1986) are examples of such drugs and
their action during sepsis will be discussed in the following Chapters of
this thesis.
Surgery
Although surgery itself is immuno-suppressive, delayed type
hypersensitivity responses can be improved by the drainage of visceral
abscesses (Meakins et al , 1979). In addition, the drainage of an intraabdominal abscess can also restore the function of 'failed' organs (Polk
& Shields, 1977), so that organ failure should be regarded as a
potentially reversible 'organ dysfunction' (Bohnen et al, 1983). However,
the use of percutaneous drainage under computerized tomography
guidance may be a reasonable choice, as the risks of a negative
laparotomy in multiple organ failure require clarification.
Nutrition
Refeeding via oral or intravenous route has been shown valuable in
correcting several abnormalities in immunological function. Total
parenteral nutrition has been reported as capable of restoring skin test
reactivity, restoring lymphocyte transformation, correcting serum
immunoglobulin levels and restoring specific antibody responses and
neutrophil chemotaxis (Nohr & Meakins, 1985). However, the choice of
the best method of nutritional support, wether by the use of enteral or
parenteal nutrition, is still controversial. The best nutritional regimen and
the timing of feeding after injury or the establishment of sepsis are also
to be defined. Despite the beneficial effects, rigorous documentation of a
survival benefit due to nutritional support is still not available.
CHAPTER 1
INTRODUCTION
Sepsis remains an important cause of death and morbidity (Altemeier,
1980; Meakins, 1985). It frequently complicates surgical operations and delays
the resolution of major illness (Cerra, 1982; McGowan & Gorbach, 1983;
Wilmore et al , 1983; Meakins, 1985). Infection occurs despite surgical advances
supported by modern and safe anaesthesia, potent and specific antibiotics, better
monitoring of the clinical and metabolic course, effective nutritional support, and
a more clear understanding and application of the principles of physiology.
The general term 'sepsis' has caused considerable confusion in clinical and
experimental studies (Wichterman et al , 1980) because it refers to different
conditions in different studies. Some experimental septic models induce a lowgrade "smoldering" infection which can last for days or weeks, whereas other
models describe a very aggressive process that causes death within hours. While
infection is a localized focus of invading mircroorganisms confined to a tissue
space, and microorganisms within the blood stream are objective evidence of
systemic invasion, a septic state can be regarded as the addition of the local and
the systemic effects produced by a focus of infection. For the purpose of the work
described in this thesis, 'sepsis' is regarded as an acute invasive infection, which
involves stimulation of phagocytosis with mobilization and activation of white
blood cells, associated with
important changes in intermediate metabolism,
causing the development of a toxic state (weakness, anorexia, pile erection,
lethargy, etc.), leading to the eventual failure of most organs and/or circulatory
collapse (septic shock).
A wide range of microbial organisms can cause sepsis, especially grampositive and gram-negative bacteria. While the term 'bacteraemia' refers to
bacteria in the blood, irrespective of whether they do or do not induce the toxic
state of sepsis, 'septicaemia' refers to a toxic state produced by viable proliferating
bacteria in the blood.
Infection in the critically ill patient can be classified into two main types,
according to the nature of the contaminating flora and some clinical
characteristics, as described by Meakins (1985):
Type 1. Usually monomicrobial
Monomicrobial infection occurs when an organ or structure becomes
infected with a single strain of organism, for example, streptococcal endocarditis,
lobar pneumonia, tuberculosis, etc. It can also occur associated with therapeutic
manipulation, for example, line infection, urinary tract infections, etc.
Type 2. Usually polymicrobial
Polymicrobial infection, in contrast, occurs when a body space becomes
contaminated with normal intestinal flora, for example, diverticulitis, perforated
colon, etc. It can be related to the surgical procedure required to manage the
primary disease, or to extrinsic complications from surgery, such as peritonitis,
empyema, enteric fistulae. It can also occur as a result of civilian or military
injuries.
FACTORS DETERMINING THE DEVELOPMENT INFECTION
The development of infection depends on three major determinants: the
causative organism, for example, the bacteria; the environmental factors; and the
general defence mechanisms, which are designed to contain and resolve the
established infection. Bacterial factors involve virulence, pathogenicity, number
of microorganisms and antibiotic resistance. Enviromental factors are the changes
that influence the milieu, including the contact with the infectious source, and the
local barriers to colonization, which lead eventually to tissue invasion. Defence
mechanisms involve the systemic response to the bacterial invasion of tissues,
including changes in the immune system, which are associated with the
physiological and metabolic response (Meakins, 1985). Fig. 1.1. shows the
interrelation of these determinants in the normal subject and in the septic patient.
Fig. 1.1.
These determinants are basic to the generation of sepsis. In the homeostatic or
normal state (Fig. 1.1. - top illustration), the intersection of the quadrangles
represents the relationship between the determinants and the probability of
infection, which is very low indeed. However, all three determinants alter
their relationship in
patients at high risk of developing infection and in the already infected patient, as
represented by the area of intersection in the bottom illustration (Fig. 1.1.).
FACTORS INCREASING SUSCEPTIBILITY TO INFECTION
Some of the environmental and genetic factors play an important role in
determining susceptibility to infection, as well as its severity.
Age
Age influences not only susceptibility to infection, but also its clinical
course. Many infectious diseases show a characteristic age distribution. Infections
with ubiquitous organisms are seen firstly in infants, shortly after they have lost
the maternal antibody transferred across the placenta, and have a peak incidence
in early childhood. Maturation of the immunological system, associated with
gradual acquisition of protective immunity due to successive exposures to
infection, appear to promote a more adapted response to infectious threats in
adulthood. With old age immunity declines increasing susceptibility to infection
again.
Sex and hormonal factors
Not only hormonal but social factors are implicated in the variation in the
sex distribution of infectious diseases. Sex has a role in defining occupation and,
therefore, can influence how close men and women come into contact with the
source of infection. Pregnancy can predispose to various infections, including
hepatitis, pneumoccocal infections, amaebiasis and malaria, possibly reflecting
changes in immunological function and in metabolism that occur at that time
(Greenwood , 1983).
The presence of conditions leading to excess glucocorticosteroids, such as
adrenal hyperplasia, adrenal tumors, or steroid therapy, may also increase
susceptibility to infections, for example, infections with herpes simplex, and
herpes zoster. Corticosteroids probably exert their action via the immune and
metabolic responses (Karakusis, 1986). Patients with diabetes, especially if poorly
controlled, show increased susceptibility to infections, with the common
development of abscesses, boils and urinary tract infections. Diabetic patients also
present with impaired cell-mediated immune reactions and chemotaxis by
polymorphonuclear leucocytes (Bagdada et al , 1974; Greenwood, 1983).
The relation of infection and/or injury with malnutrition and immunity
A number of clinical and experimental studies have shown that
defficiency of protein, of individual vitamins, and of trace elements increase
susceptibility to infection (Chandra, 1979; Hessov, 1981). The work with protein
energy malnutrition in developing countries, where many children suffer from
undernutrition and frequently die from infection, has revealed a close relationship
between nutritional status, immune response and infection ( Chandra &
Newbourne, 1977). The clear association of nutrition and infection could be seen
recently during the famine in Ethiopia and other African countries which led to
wide-spread profound malnutrition as the main predisposing factor in the death by
infection of tens of thousands of people (Alexander, 1986). In industrialized
countries, severe malnutrition is encountered as a complication of other
conditions, such as generalized malignant disease, cirrhosis, and intestinal
malabsortion due to inflammatory bowel disease, more frequently than as a result
of pure dietary deficiency. However, considerable attention has been given to the
fact that malnutrition is also widely prevalent in surgical patients, and bears a
close association with morbidity and even mortality (Bistrian et al , 1974; Hill et
al , 1977; Hessov, 1981). Spanier et al (1976), in body composition studies,
directly related the depletion of body cell mass to immunocompetence and
repletion of body cell mass with restoration of immune function. Malnourished
patients may present delayed hypersensitivity (Meakins et al , 1977). Anergy
occurs in prolonged starvation and appears to be reversed by refeeding (Spanier et
al , 1976). However, the relationship between nutritional state and
immunocompetence is far more complex, for other factors beside malnutrition,
such as injury, surgery and infection itself, may also intervene in the imunne
response (Hill, 1981). Infection and/or injury may in turn contribute to the
protein-calorie malnutrition, possibly by causing anorexia and simultaneously
increasing the energy requirements. It is, therefore, probable that the
malnourished patient who becomes infected, with or without associated injury,
enters a vicious circle with a potential spiral descent to innanition, anergy and
death due to overwhelming infection (Fig. 1.2.) (Kettlewell et al , 1979).
Fig. 1.2.
In his classical metabolic balance studies, in patients with long bone
fractures, Cuthbertson (1930) emphasized the significance of proper nutrition in
reducing the protein loss following injury or surgery. Today, careful assessment
of the nutritional status leading to administration of nutritional support to the
malnourished hospitalized patient, is well incorporated into modern clinical care
practice (Dudrick et al , 1971; Elwyn, 1980; Kettlewell, 1982; Winters & Greene,
1983; Silk, 1983; Phillips & Odgers, 1986).
Some studies have reported that the survival of some groups of patients,
such as those with intestinal fistulae, severe sepsis, multiple injuries and after
radical oesophagogastric surgery has improved in recent years, and this has been
attributed, in part, to the role of nutritional support, either as enteral or parenteral
nutrition (Elwyn, 1980; Mullen et al , 1980; Yamada et al , 1983; Philips &
Odgers, 1986).
Drugs
Malnutrition and infection in cancer patients receiving cytotoxic or
immunosupressive drugs is well recognized, and has led to the use of intravenous
feeding as adjuvant therapy (Fischer, 1984; Copeland, 1986). Alcohol, in excess,
increases susceptibility to many infections, especially when liver failure
supervenes. Among other factors decreasing the resistance of alcoholics to
infection is impaired cell mediated immunity, in particular reduced mobilization
of granulocytes and impaired clearing function of the macrophage- monocyte
system, loss of delayed hypersensitivity and a decrease in t lymphocytes in
peripheral blood. (Lieber & DeCarli, 1977). The neglect of simple hygienic
precautions when administering their intravenous injections in hard-line drug
addicts leads to septicaemia as a common terminal event, or alternatively, to the
contamination with the Acquired Immunodeficiency
Syndrome
virus (Anon,
1986).
Smoking
also predisposes to respiratory infection due to the damage of
the epitheleum of the respiratory tract (Greenwood, 1983) .
Malignant disease
Immune responsiveness is impaired in patients with dessiminated
malignant disease (Kettlewell, 1979). The frequent association of cancer and
malnutrition, added to the harmful effects of radiotherapy or treatment with
cytotoxic drugs, further increases the risk of developing infection in these
patients. The provision of nutritional support, with the aim to correct malnutrition,
has been largely used in such patients, although whether this contributes to
diminishing the infection risk is still to be established clearly (Fischer, 1984;
Copeland, 1986).
HISTORICAL ASPECTS AND THE IMPORTANCE OF INFECTION
Infection was the major factor in worldwide population control of
mankind until very recently. Before the 17th century, epidemics of smallpox,
plague diphtheria, measles, cholera and infectious diarrhoeal diseases, were
common episodic events causing death of more than 50% and sometimes as much
as 90% within a region (McNeil, 1977). Better hygiene and improved nutrition
increased the average life expectancy, and contributed to the five-fold increase in
the world population over the last 150 years (Alexander, 1986).
Over this period, the explosion in scientific methods, associated with the
constant search for controlling and understanding the mechanisms of infection,
have had a tremendous effect on infectious diseases. Smallpox, one of the most
common causes of death in the world a few hundred years ago, is now extinct
(Barnes & Robertson, 1981). By contrast, the control and eventual cure of the
potentially catastrophic Acquired Immuno Defficiency Syndrome, caused by an
infection with the human immuno-deficiency virus, poses a similar challenge to
the scientific community today (Anon, 1986).
Infection may be part of a primary disease process, for example
pneumonia, or may occur as a complication of treatment, for example pelvic
abscess following abdominal surgery or line infection during intravenous feeding.
However, until the 19th century, the formation of pus was considered a normal
part of healing - pus bonum et laudbile . John Hunter [1728-1793] recorded in
his "Treatise on the Blood, Inflamation and Gun Shot Wounds", published in
1794, that "inflamation is not only the cause of diseases but it is often the mode of
cure". Louis Pasteur [1822-1825] caused a dramatic change in the surgical
attitude towards infection by showing that fermentation was caused by living
multiplying matter and, therefore, that pus formation had to be also caused by
minute organisms from the enviroment. Joseph Lister [1827-1912] by the use of
carbolic established the principles of antisepsis and revolutionized surgical
practice. This major revolution in surgery, which progressed to the use of aseptic
surgical technique, led to a great reduction in surgical morbidity and mortality.
Lister's first scientific analysis of the septic state is found in his "On the early
stages of inflammation" (1858) when he thought "The effects of irritation of the
tissues are twofold. Firstly, there is a dilatation of the arteries which is produced
through the nervous system. Secondly, there is an alteration in the tissues on
which the irritant acts directly. This alteration imparted an adhesiveness to both
the red and the white corpuscles, making them prone to stick to one another and
to the wall of the vessels, and so giving rise to stagnation of blood and ultimately
to obstruction" (Singer & Underwood, 1962).
Since Thomas Latta (1832) first reported in the Lancet the prevention of
death from cholera by the intravenous injection of 6 pints of almost normal saline
containing 3 drachms of salt and 2 scruples of sodium bicarbonate, or about 2.8
litres of 89 mM Na+, 78 mM Cl-, and 11 mM bicarbonate, the use of fluid and
electrolyte therapy has become such a familiar part of medicine that it is rarely
considered today (Veech R L, 1986). The successful clinical use of antibiotics,
described for the first time by the Oxford group (Abraham et al , 1941) was a
gigantic step in the fight against infection.
However, the introduction of asepsis and antisepsis, fluid and electrolyte
therapy, specific antibiotics, careful monitoring, aggressive operative intervention
and nutritional support, has not eliminated infection as an important clinical
problem today. Studies have indicated that approximately one third of surgical
patients suffer from infection at some stage of their stay in hospital. Around 10%
of operated patients may develop wound infections, which will lengthen the
hospital stay by a week on average (Altemier et al , 1976; Green et al , 1977;
Altemier, 1980; Cruse & Foord, 1980). Surgical sepsis plays an important role in
the morbidity and mortality encountered in intensive care units and constitutes a
major impediment to the resolution of critical illness, with septic patients
presenting a three-fold mortality rate as compared with non-septic patients
(McLean & Boulanger, 1985). The increasing use of immunosuppressive drugs
and corticosteroids increases the susceptibility to infection (Cupps & Fauci, 1982;
Greenwood, 1983; Copeland, 1986). More severely injured patients, transplant
patients, patients with cancer and diabetes melitus are surviving longer, so
increasing the number of patients at increased risk from infection. In addition,
invasive techniques such as indwelling intravenous catheters, especially longterm catheters used for intravenous feeding, and bladder catheters also increase
the risk of sepsis (Platt et al , 1982; Pinilla et al , 1983).
The magnitude of the stress imposed by sepsis is a function of the size of
the infective process, the number of invading organisms and their virulence.
Survival depends not only on the adequacy of the immunological reactions to
contain and eliminate invading microbes, but also on a series of integrated
physiological and metabolic responses necessary to maintain energy production
and cell function thoughout the body (Clowes et al , 1985). It is encouraging,
however, that the complex pathophysiology of bacterial sepsis is gradually
becoming better understood.
THE METABOLIC EFFECTS OF STARVATION AND INJURY
Sepsis is frequently associated with starvation and/or injury. A summary
of the metabolic effects brought about by these latter conditions is shown in Fig.
1.3.
Fig. 1.3 .
Metabolic changes in starvation
In the transition from the fed to the starved state, a sequence of metabolic
alterations occurs as the body maintains glucose homeostasis in the early phase,
and then preserves body protein mass in the later phases of starvation. In early
starvation, glycogen stores
are largely exhausted within 24 h, and to avoid hypoglycaemia and
maintain the demand for glucose by the brain, red blood cells and renal medulla,
gluconeogenesis is activated. As a result of diminished or abolished absorption of
glucose from the gut, plasma insulin concentrations fall enabling the increased
release of amino acids (alanine and glutamine) from protein, to be used mainly as
gluconeogenic precursors. The low concentrations of insulin also allow the
release of free fatty acids from adipocytes to meet the major portion of the body's
energy requirements, either through their total oxidation, or via the increased
utilization of their partial oxidation products, namely, ketone bodies. The elevated
rate of protein degradation, necessary to provide precursors for the increased
gluconeogenic demand, is progressively reduced as starvation proceeds, leading
to a decrease in the basal metabolic rate. A major factor in this protein-sparing
adaptation, essential for survival during prolonged periods of starvation, is the
gradual change from oxidation of glucose to oxidation of ketone bodies by tissues
such as the brain, which normally metabolizes glucose when ketone bodies are
not available. A more detailed discussion of the metabolic adaptation to starvation
can be found in Felig et al (1969), Cahill (1976), and Newsholme & Leech
(1983).
Metabolic changes in injury
Whereas the metabolic changes in starvation are geared towards
conserving energy output, the metabolic response to injury is directed towards
coping with the increased energy demands, and to the support of the healing
wound. These responses are proportional to the severity of the injury and are
characterised by three distinct phases (Fig. 1.4.): the early ebb or shock phase,
the subsequent flow or hypermetabolic phase, and the recovery or anabolic phase
(Cuthbertson, 1930). The changes presented in these phases have many
similarities when compared to those found in the response to sepsis. (For review
see: Silk, 1983; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986).
Fig. 1.4.
The ebb phase
The ebb phase is characterized by a rapid fuel mobilization,
haemodynamic instability and increased plasma catecholamine concentrations
(Coward et al , 1966). The metabolic rate falls during this phase (O'Donnell,
1976). Insulin plasma concentration is variable (Frayn, 1986) despite a possible
inhibition of its release by the action of the higher catecholamines on the
pancreatic ß-cell (Porte & Robertson, 1973). Glucocorticoids and glucagon are
also reported to be elevated (Forse & Kinney, 1985). Glycogen and
triacylglycerol stores are mobilised for immediate energy production, although
there is some argument about the need, and how adaptive such a massive fuel
mobilization is (Frayn, 1986). Maintenance of circulatory volume and tissue
perfusion appear to be aim during this phase.
The flow phase
If death does not occur as a result of hypovolaemia or direct damage to
vital organs during the ebb phase, a more prolonged period follows (the flow
phase), which is characterized by an increase in the
metabolic rate, breakdown of body tissue and weight loss. Increased plasma
concentrations of insulin and glucagon have been reported during this phase
(Biesel & Wannemacher, 1980; Neufeld et al , 1982). Generally this phase of the
metabolic response can be regarded as a balance between the action of insulin and
those of the counterregulatory hormones, namely, glucocorticoids, glucagon and
catecholamines (Forse & Kinney, 1985). Another characteristic feature of this
phase is the presence of hypermetabolism (Cuthbertson, 1979), which appears to
be proportional to the degree of stress (Elwyn et al , 1981). The function of this
hypermetabolic catabolic phase appears to be to maintain energy production and
cellular activity thoughout the body, at a time dietary intake may be scarce and/or
not absorbable. If this is achieved and the immunological and metabolic responses
take a successful course, the changes leading to net catabolism are gradually
replaced by those leading to net anabolism, merging into the 'anabolic' or
'convalescent' phase.
The anabolic phase
The transition from the catabolic flow phase to the anabolic period of
convalesce can occur quickly, for example within a few days, as with soft tissue
injury, or can occur over several days as with extensive trauma (Moore &
Brennan, 1975). The distinct metabolic changes that occur during anabolism aim
to replace the tissue lost over the catabolic phase, whereas resting energy
expenditure may fall from the elevated levels of the flow phase to subnormal
levels, similar to those found in depleted patients (Moore & Brennan, 1975; Forse
& Kinney, 1985).
GENERAL DESCRIPTION OF THE METABOLIC RESPONSE TO SEPSIS
The metabolic response to sepsis is not only directed to maintainance of
energy production and cellular function thoughout the body (Clowes et al , 1985),
but also to the containment and resolution of the established infection.
Many of the changes in the metabolic response to injury are shared by the
metabolic response to sepsis. However, while the pattern of the response to injury
shows a clear time-sequence, from the moment of trauma until recovery, the
course of the response to sepsis is less predictable, because of fluctuations in the
nature and intensity of the metabolic changes, which in turn, are linked to the
severity and type of infection, variations in the nutritional and hormonal state, or
failure in the defence mechanisms. Take, for example a patient with intraabdominal sepsis proceeding well during the flow phase. If part of the avascular
fibrinous wall of a previously isolated-off abscess is eroded bacteraemia can
develop, which in turn, will precipitate events of the ebb phase, which in turn,
may lead to 'septic shock'.
The ebb phase of injury/trauma may be different from that of sepsis. In the
former, there is decreased energy production associated with hypovolaemia,
whereas in the ebb phase of sepsis, the patient in 'septic shock' may present in a
hyperdynamic state, with increased energy production, hyperventilation,
increased central venous pressure, increased cardiac output, oliguria, decreased
peripheral resistance, hypotension and lactic acidosis (Siegel et al , 1967; Forse
& Kinney, 1985). This hyperdynamic septic state then evolves to the
hypodynamic septic state with low central venous pressure, hypotention, low
cardiac output and increased peripheral resistance (MacLean, 1981; Forse &
Kinney, 1985). This suppports the view of a bimodal response to severe acute
sepsis, with an initial hypermetabolic hyperdynamic period followed by a
hypometabolic hypodynamic late septic state (Chaudry et al , 1979; Wichterman
et al , 1980).
The hypermetabolism of the flow phase of sepsis, as in that of injury,
appears to be proportional to the severity of infection, so that during major
infections, such as peritonitis, there may be increases of up to 60% of the resting
energy expenditure (Elwyn, 1980). It is during this phase that the septic process
appears to enhance a progressive inability of insulin to promote its anabolic
actions, leading sometimes to the development of hyperglycaemia and glucose
intolerance (Clemens et al , 1982). The hormonal changes include increases in
insulin and glucagon and glucocorticosteroids (Beisel & Wannemacher, 1980).
Amino acid catabolism is enhanced, as well, as the formation of lactate, alanine
and glutamine in muscle (O'Donnell et al , 1976; Hasselgren, 1986). The efflux of
substrates from muscle to the liver associated with the hormonal changes
promotes alterations in hepatic glucose and fat metabolism. Part of the glutamine
released from muscle enters the kidney where it is converted to glucose and
ammonia (produced to enable sufficient protons to be excreted and so prevent
acidosis), part is transformed into alanine in the intestine, and part contributes to
the synthesis of acute phase proteins, necessary for host resistance (Phillips &
Odgers, 1986). The increased utilization of aminoacids in the liver leads to
increased hepatic protein synthesis (Hasselgren et al, 1984), and to increased
ureagenesis (Beisel & Wannemacher, 1980), especially from those aminoacids
that cannot be catabolised in muscle (e.g. phenylalanine and tryptophan). The
mobilization of non-esterified fatty acids is variable, however net whole body
utilization of fat continues during the septic process as judged by measurements
using indirect calorimetry (Askanasi et al , 1979). The elevation of ketone bodies
during starvation is inhibited by sepsis (Neufeld et al , 1982). Hepatic function is
altered (Royle & Kettlewell, 1980) and lipid accumulation in the liver may occur
(Guckian et al , 1973; Champion, 1976). Eventually, the metabolic alterations
may lead to multiple organ failure and death (Duff, 1985).
However, the reason for the proteolysis, the regulation of the changes in
hepatic metabolism, and the mechanism of development of the multiple organ
failure syndrome are still not completely understood. Two interdependent
hypotheses, namely "substrate deficiency" and "endocrine activation" have been
put foward to attempt to explain the catabolism of injury/sepsis. In the former, the
increased metabolic requirements would be met be proteolysis, lipolysis, and
gluconeogenesis. In the endocrine activation hypothesis, the increased production
of hormones, such as catecholamines, glucagon and corticosteroids, would lead to
the catabolism of injury/sepsis (Moore, 1983).
Among many of the metabolic studies of sepsis, there is usually variability
in the severity and type of the septic insult, which is often associated with
variable degrees of trauma, the latter being commonly accompanied by different
nutritional and hormonal status. Furthermore, it is also not unusual to find
variation in the time the observations are made; and time plays a very important
role in the progress and nature of the metabolic changes brought about by sepsis.
Interpretation or comparison of the results under these circunstances, therefore,
may be misleading, for they may reflect, in part, the effects of the different
experimental conditions. This may account for some of the apparent discrepancies
found in the published reviews, in both experimental and clinical sepsis
(Blackburn, 1977; Beisel & Wannemacher, 1980; Cerra, 1982; Neufeld et al ,
1982; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986), as illustrated by
Fig. 1.5.
Fig. 1.5.
More detailed discussion of changes in metabolism of carbohydrate and
fat in response to sepsis will be included in the experimental Chapters.
MULTIPLE ORGAN FAILURE
The failure of vital organs in the septic and/or injured patient has received
the attention of a number of recent studies (for review see: Polk, 1982; Duff,
1985). It is now clear that several organs are often involved, usually remote from
the site of infection. In addition, the clinical manifestations of their involvement
show a sequential pattern. Failure of the renal, respiratory and cardiovascular
systems often occur first, and, if this is not followed by recovery, there is failure
of the brain, liver and gut (Duff, 1985).
The lungs
The earliest recognized form of organ failure was that involving the lung.
Adult respiratory distress syndrome (ARDS), 'shock lung', 'Viet-Nam lung' and
'lung lesion of sepsis' all describe a type of respiratory failure, with each name
being related with their initiating event: shock, trauma and/or sepsis (Polk, 1982).
The predominant characteristic of adult respiratory distress syndrome appears to
be the association with infection ( Horovitz & Shires, 1974; Eiseman & Narton,
1975; Fulton & Jones, 1975). The pathophysiology of lung failure is not clear, but
there is some evidence from clinical and experimental studies that the pulmonary
oedema is a result of increased permeability of pulmonary capillaries. Increased
capillary permeability has been reported in sheep injected with endotoxin or
bacteria, as judged by the increase in the rate of appearance and the concentration
of protein in the lung lymph (Brigham et al , 1979). Adult patients with ARDS
showed increased rate of incorporation of radiolabelled plasma albumin into the
bronchoalveolar fluid (Sibbald et al , 1979). The mechanism of capillary injury
is not fully explained, but the effects of complement activation may play an
important role (Craddock et al , 1977; Jacob et al , 1980). This endothelial cell
injury could also be the result of the action of the polymorphonuclear leucocytes
(or their products) which are known to be aggregated by activated complement.
To support this view there is evidence that drug-induced neutropoenia can
diminish or prevent the effects of infusion of activated complement (Craddock et
al, 1977). Other causative factors are also likely to be involved, as experimental
lung lesions caused by infusion of complement are never as extensive as those
observed in septic patients (Hyers, 1981). Using a radioaerosol lung scanning
technique, Tennemberg et al (1987) studied patients with trauma and sepsis, and
reached the conclusion that complement-mediated neutrophil activation, alone,
appeared to be unable of initiating lung injury or precipitating ARDS.
Complement-mediated neutrophil activation may, therefore, serve as an early,
potentiating mechanism for other polymorphonuclear triggering mediators. It is
also well known that the lungs, in addition to their gas exchange and filter
function, and the fact they are target-organs of circulatory factors, they clear
many substances from the circulation such as 5-hydroxytryptamine. The lungs
also synthesise and release various agents, that may act on smooth muscle and,
therefore, interfere with the function of other organs; the lungs being thus
regarded in a way analogous to an "endocrine gland" (Aun & Birolini, 1985).
The kidneys
The pathophysiology of the injuries leading to renal failure is not fully
understood. Acute tubular necrosis may or may not be present. Decreased renal
blood flow, endotoxin action and vasoconstriction of renal arteries have been
implicated as possible mechanisms (Duff, 1985). Fortunately renal failure can be
treated successfully with haemodialysis until function returns, but the latter is
usually linked to the recovery of function of other organs, especially the liver.
The liver
Liver function is almost always impaired in sepsis whether accompanied
by the multiorgan failure syndrome or not. Liver function tests show a pattern
suggestive of cholestasis with a rise in serum bilirubin and alkaline phosphatase,
accompanied by moderate elevation of SGPO and SGPT (Royle & Kettlewell,
1980; Duff, 1985). Jaundice is the clinical sign which reflects such changes, and
appears at a later stage in the septic process, as compared to the biochemical
changes. Changes in blood amino acid concentrations include elevations of
phenylalanine, taurine, cystine and methionine, which are metabolized in the liver
(Freund et al , 1978). Macroscopic changes in livers of monkeys, injected with
live E. coli bacteria, include intravascular sequestration of degranulating
leucocytes and fibrinous deposits of platelet aggregates in sinusoids and the
spaces of Disse (Balis et al , 1979). The post-morten of patients who died with
multi-organ failure reveals hepatic changes such as, focal necrosis, fat
accumulation, congestion and enlargement of sinusoids, and cholestasis
(Champion, 1976).
Gastrointestinal system
The gastrointestinal (G.I.) system may fail due to ulceration, loss of
barrier function or malabsorption. In patients dying from multi-organ failure, the
post-morten shows massive denudation of the gastrointestinal tract mucosa, which
suggests a failure in the gastrointestinal barrirer function, enhancing abnormal
absorption of bacteria and/or bacterial products (toxins) (Duff, 1978 and 1985).
Pathophysiological mechanisms of multiorgan failure syndrome
A common mechanism used to explain the development of multiorgan
failure is based on the action of free radicals (O2-, OH-, H2O2). As a result of
sepsis, or sepsis associated with trauma (usually an operation to treat localized
infection), there is activation of the complement cascade through the alternative
pathway, and release of factor C5a (a polypeptide of 74 residues and a molecular
weight of 15,000). This peptide interacts with circulating neutrophils to increase
their adherence and potential to degranulate when exposed to suitable stimuli
(Simmons & Solomkin, 1985). The aggregation of granulocytes, in turn, releases
free radicals in amounts sufficient to cause cellular damage, dysfunction and
eventualy organ failure (Duff, 1985).
Recent evidence, however, appears to indicate a very important role of the
macrophage in the response to sepsis. Macrophages activated by bacteria or
bacterial products, such as endotoxin [lipopolysaccharide (LPS)], can release a
polypeptide "hormone" called Cachectin which might initiate tissue injuries,
which are the same as those caused by endotoxin inoculation (Tracey et al ,
1986; Beutler et al , 1987).
SEPSIS AND ENDOTOXIN
Endotoxin is a lipoprotein-carbohydrate complex (lipopolysaccharide LPS) found in the cell wall of all gram-negative bacteria. Andre Boivin (1941)
was the first to isolate endotoxins from gram-negative bacteria, and some ten
years latter endotoxins were implicated as the cause of human gram-negative
shock (Borden & Hall, 1951; Braude, 1953). This hypothesis has led to numerous
studies on the physiological and biochemical alterations associated with
endotoxin inocculation into various laboratory animals over the last four decades.
Furthermore, some of the results of such studies are
usually incorporated into the descriptions of the pathophysiology of clinical sepsis
and septic shock, and in their treatment (Wichterman et al , 1980).
Very few septic patients have endotoxin identified in their blood, and if
they do, the concentrations are quite low (Levin et al , 1972; Postel et al , 1975).
Recent studies, however, support the view that endotoxin acts indirectly, via
stimulation of polymorphonuclear leucocytes (Tracey et al , 1986; Beutler &
Cerami, 1987; Christou et al , 1987).
There are differences in some of the clinical events that result from
sepsis as compared to those induced by endotoxin inoculation (Fig. 1.6.), as
described by Wichterman et al (1980).
Fig. 1.6.
Nevertheless, both endotoxin and sepsis can activate the macrophages to
produce interleukin-1 and/or its by products, which appear to be important
mediators of the metabolic changes brought about by sepsis and/or endotoxin
(Cohn, 1978; Clowes et al , 1983). Both sepsis and endotoxin can also cause
similar hormonal and metabolic changes, such as, hyperinsulinaemia and
hyperglucagonaemia (Yelich & Filkins, 1980; Beisel & Wannemacher, 1980),
and the inhibition of the hyperketonaemia in response to starvation (Neufeld et al
, 1982). The development of multiple organ failure syndrome also appears to
follow both endotoxaemia (Tracey et al , 1986) and sepsis (Duff, 1985). These
findings suggest the metabolic response to LPS is similar to that of sepsis,
although variation in the time-course of the resulting clinical events may occur.
These differences may be due to the endotoxin dosage, or to the time necessary to
activate the immunological cascade by endotoxin inoculation, as compared to that
with the various ways clinical sepsis behaves. Therefore, data concerning
endotoxin treament, whenever considered relevant, will be included in this
Thesis.
THE EFFECT OF GRAM-POSITIVE BACTERIAL COMPONENTS DURING
INFECTION
The
major
irritative
components
of
gram-positive
bacteria
are
peptidoglycan and teichoic acid. Peptidoglycan is efficient at causing an abscess:
10µg in the skin of man is an adequate dose (Murphy, 1983). It appears to be
unable to fix complement via the alternative pathway. However, since all normal
sera contain anti-bodies against peptidoglycan, it does fix complement by the
classical pathway. Peptidoglycan is able to activate macrophages, but
concentrations of 10-100 ng/ml are required, as compared to the concentration of
1ng/ml required for maximal stimulation by endotoxin. The active component
appears to be N-acetyl muramyl-lala D isoglutamine (MDP), which is about 100
times more active than peptidoglycan when coupled to an inert carrier.
Macrophages activated by MDP secrete the same substances as the macrophages
stimulated by endotoxin. Techoic acids are efficient activators of complement via
both the alternative and classical pathways.
THE ROLE OF PHAGOCYTIC CELLS IN INFECTION
What happens during an infection is a function of how far the invading
organisms are successful in multiplying. Single organisms are disposed of by
phagocytosis, without any further response. Even a few thousand bacteria can
cause microscopic inflammation. A few million organism induce the generation
of substances which cause enough vasodilataion, increased capillary permeability,
and chemotaxis to be clinically visible as inflammation. Many millions of
organisms are required to cause a local abscess. Tissue destruction is actually
caused by proteolytic enzymes liberated from dead and dying phagocytic cells.
Phagocytic cells, including neutrophils, monocytes and macrophages, are
responsible for the process of phagocytosis. At the beginning of this century
Metchnikoff (1905) described phagocytosis as the ingestion and subsequent
killing of micro-organisms (endocytosis), and believed this was a very important
process in both the prevention and fight against infection. Latter on, however, it
became clear that an important contribution of phagocytes to the inflamatory
response is the exocytosis of enzymes and other proteins, some of them possibly
carriers of important signals for the development of this response (inflammatory
or humoral mediators). Exocytosis, on the other hand, can occur independently
from endocytosis and in response to different stimuli from those of the latter
(Bentwood & Henson, 1980).
These proteins released by the inflammatory cells are capable of various
actions, such as making the site of inflammation accessible to an adjacent
vascular space (e.g. collagenase and elastase); regulating local inflammatory
mediators (e.g. carboxypeptidase); controlling the concentration of metabolites
involved in bacterial growth (e.g. lactoferrin and vitamin B12); and enhancing an
extracelluar environment adverse to microorganisms through the action of acid
hydrolases, cationic proteins and lysozymes (Simmons & Solomkin, 1985).
THE ROLE OF THE MACROPHAGE DURING SEPSIS
In bacterial infections, cell wall polymers are recognized as foreign by
processes which do not require antibody. In viral infections, the initial insult is
usually the death of infected cells, which liberate endogenous inflammatory
mediators. Immune responses, mediated either by antigen-antibody complexes or
by sentitized lymphocytes become increasingly important in the inflammatory
response to chronic infection. Each of these mechanisms activates the same
enzymatic cascades; the complement, kinin, coagulation and fibrinolytic products.
The crucial cell which is activated appears to be the macrophage.
Macrophages are distributed throughout the body and have developed
specific characteristics in various organs, from which they acquired their
respective names (Kuppfer's cells within the liver, alveolar macrophages in the
lung, mesangial cells in the kidney, and glial cells (which are thought to be
derived from macrophages) in the brain. It appears to be clear that macrophages
have adapted for local defence purposes, and in addition, macrophage function
can also vary at different sites (For review sse: Takemura & Werb, 1984). The
three primary functions of macrophages, described by Cristou et al (1987), are
listed bellow:
1. Immune function
Antigen presentation
2. Phagocytic-microbicidal function
Phagocytosis
Destruction of viral infected cells
Oxidative killing mechanisms
Chemotactic factors
Lysozyme release
Destruction of intracellular parasites
3. Secretory function
Proteases
Procoagulant activity
Interleukin 1 (IL-1)
Colony stimulating factor
Angiogenesis factor
Plasminogen activating factor
Prostaglandins
Leukotrienes
Interferon
Tumor necrosis factor (TNF)/Cachectin
The differences in macrophage function could also be understood in terms
of degree of macrophage activation, namely, how 'stimulated' the macrophage is.
A spectrum of stimulation can be recognized depending on the potency of the
stimulating agent. Thus, macrophages which are stimulated with e.g. interferon or
complement are refered to as "inflammatory" or 'elicited" macrophages, whereas
macrophages stimulated, for example, by endotoxin (Lipopolysaccharide - LPS)
are fully "activated" macrophages (Cohn, 1978). The activation of macrophages
results in enhanced phagocytosis (Cohn, 1978), increased production of toxic
oxygen radicals (Tsunawaki et al , 1974) and the release of various proteases and
hydrolytic enzymes (Takemura & Werb, 1984).
There is also release (Fig. 1.7) of various monokines (Takemura & Werb,
1984; Dinarello et al , 1986;
Kovaks et al , 1985).
Fig. 1.7.
Aggarwal et al , 1985; Beutler et al , 1986;
Release of oxygen radicals by activated macrophages (Babion et al,
1973; Craddock et al, 1977) has also been reported in sepsis.Interleukin 1 can be
produced by virtually all activated macrophages (Dinarello & Mier, 1986) and has
been implicated as an important mediator in the hypercatabolism of proteins in
response to sepsis (Baraco et al, 1983; Clowes et al, 1983 and 1985).
Macrophages treated with endotoxin (LPS), in vitro , show altered secretion of
various proteins (Largen et al, 1986). An important monokine released by
endotoxin stimulation of macrophages is the Tumor Necrosis Factor (Cachectin),
which appears to be an important humoral mediator of the systemic metabolic
changes during sepsis (Butler et al, 1985; Cerami et al, 1986). Studies using coculture of hepatocytes with Kupffer's cells or macrophages, in the presence or
absence of inflammatory stimulation, showed that there was a significant
depression of hepatocytes' protein synthesis when endotoxin (LPS)
was added to the co-culture medium. However, in the absence of either
macrophages or Kupffer's cells, endotoxin had no direct effect
on hepatocytes' protein synthesis (West et al , 1985 and 1985). More recent
evidence suggests that the macrophage-mediated inhibition of protein synthesis in
isolated liver cells occurs via a monokine mediator yet to be identified, but neither
Interleukin-1 or Cachectin (Christou et al ,1987).
The importance of macrophages in the response to sepsis can also be
demonstrated by the important role of resident macrophages in the peritoneal
cavity to fight bacteria and avoid peritonitis. Within 20 min of bacterial injection
into the peritoneal cavity, approximately 40% of the bacteria inoculated will be
associated with macrophages (Dunn et al , 1987). Baker and Kupper (1985) using
the caecal-ligation and puncture (19-gauge needle) model in two strains of mice,
showed 100% mortality in septic endotoxin-sensitive mice (C3H/HeN), whereas
the mortality of septic endotoxin-insensitive mice (C3H/HeJ) was only 25%,
within the same period of time after the induction of sepsis (4 days). These two
strains of mice differ only in one gene, which controls some aspects of
macrophage function (Adams et al , 1981). Therefore, although macrophages are
necessary for local protection against sepsis, infection-stimulated macrophage
secretory products may also be responsible for the induction of some of the
irreversible changes of the response to sepsis, for example multiple organ failure.
THE ROLE OF INTERLEUKIN-1 IN THE METABOLIC RESPONSE TO
SEPSIS
Interleukin-1 is critically important in the normal immune response by
allowing T-helper cells to differentiate and secrete Interleukin 2, which is
required for numerous lymphocyte effector functions (Fig. 1.4.). Interleukin-1, or
its by-products, have been suggested as the link between the immunological and
the metabolic response to sepsis (Clowes et al , 1986). Interleukin-1 is a
polypeptide secreted by macrophages (molecular weight 14,000 - for review see:
Dinarello & Wolff, 1982; Dinarello & Mier, 1986). Because the biochemical
nature of every macrophage-derived factor is not yet well defined, some of these
factors, initially described by their performance in different functions, may
actually be the same molecule. There is some evidence now that Interleukin-1
plays multiple roles (Takemura & Werb, 1984). It can act as a potent chemotaxin
for neutrophils and enhance endothelial adhesiveness. It has also effects on
fibroblast proliferation and may be important in wound healing (Dinarello,
1984). Endogenous pyrogen, which acts on the hypothalamus to change the
setting of body temperature (Baracos et al , 1983) and acute phase protein
synthesis promoting factor (Stein et al , 1981) all seem to be Interleukin-1.
Muscle proteolysis induced by sepsis appears to be stimulated by a circulating
cleavage product of Interleukin-1, namely, proteolysis inducing factor (Clowes et
al, 1983 and 1985). It has been suggested that leucocytic pyrogen (interleukin-1)
or proteolysis-inducing factor may act in the muscle by increasing the production
of prostaglandin E2 (PGE2) which, in turn, activates lysosomal proteases by a
calcium dependent mechanism (Rodeman et al , 1982; Freund et al , 1986).
PGE2 is derived from arachinodate via the cyclooxygenase reaction (Rodeman
et al , 1983). Therefore, cyclooxygenase inhibitors, such as indomethacin and
aspirin, might be useful in attenuating or decreasing
muscle proteolysis induced by sepsis.
Haemodynamic abnormalities and survival rates have been reported to be
improved by the use of indomethacin in various endotoxin and septic models
(Fletcher et al , 1976 and 1977; Short et al , 1981; Fletcher, 1982 and 1983).
However, administration of indomethacin to rats, subjected to caecal-ligation and
puncture, did not alter the increased muscle proteolytic rate found in these septic
rats. When indomethacin was added in vitro the release of PGE2 by septic and
normal muscles, incubated in the presence of septic plasma, was reduced by 50%,
but the increased proteolytic rate in these muscles remained unchanged
(Hasselgren et al , 1985). On the other hand, Freund et al (1986) showed that
whole body protein degradation was significantly lower in indomethacin-treated
septic rats, but this was associated with lower plasma albumin and decreased
protein synthesis by muscle and liver, at a time when new protein production is
essential. Miccolo et al (1986) using a pharmacological dose of aspirin inseptic
patients, and measuring urea production rate through the incorporation of 15N
into the plasma pool, showed no effect of aspirin on the protein breakdown rate in
septic man.
Crude leucocytic endogenous mediator (LEM) consists of substances
derived from polymorphonuclear leucocytes, which includes endogenous pyrogen
(interleukin-1), and is reponsible for inducing acute-phase reactant production in
the liver and changing the plasma concentrations of certain divalent cations,
namely, iron and zinc downwards and copper upwards (Dinarello, 1984;
Kampschmidit, 1984). LEM is also able to induce hyperinsulinaemia,
hyperglucagonaemia, glycogen depletion, and other metabolic changes similar to
those found in sepsis (George et al , 1977).
Interleukin-1, in vitro , does not affect the inhibited muscle amino acid
uptake in response to sepsis (Hassselgren et al , 1986).
However, interleukin-1 injected into the rat in vivo , had a stimulatory effect on
hepatic metabolism, in isolated liver cells, in a time-dependent pattern (Roh et al ,
1986). These changes included stimulation of gluconeogenesis from
alanine[0.5mM], elevation in non-secretory protein synthesis and increased
oxygen consumption. If interleukin-1 was added to the hepatocyte incubation
medium, however, no stimulatory effect was obtained, suggesting the effects of
interleukin-1 upon isolated hepatocytes are indirect. This view of the indirect
action of interleukin-1 in the liver, in vitro , is further supported by the fact that
interleukin-1 added to hepatocytes' culture alone, or to hepatocytes co-cultured
with Kuppfer's cells, had no effect on rates of protein synthesis (Christou et
al ,1987).
THE IMPORTANCE OF CACHECTIN DURING SEPSIS
Cachectin (or Tumor Necrosis Factor) is a polypeptide hormone secreted
by the macrophage, originally isolated in the course of studies of the mechanisms
reponsible for cachexia in chronic disease, and known for its capacity to modulate
adipocyte metabolism, to lyse tumor cells in vitro , and to induce haemorrhagic
necrosis of certain transplantable tumors in vivo (for review see: Tracey et al ,
1986; Beutler & Cerami, 1987). It has a sub-unit size of approximately 17
kilodaltons and constitutes between 1 and 2 % of the total secretory protein
produced by endotoxin-activated macrophages in vitro (Beutler et al , 1985).
The role of cachectin as a mediator of endotoxic shock was suggested by
the fact that mice passively immunized against this 'hormone' were found to be
protected against the lethal effect of endotoxin (lipopolysaccharide), as reported
by Beutler et al (1985). Tracey et al (1986) infused cachectin into rats, in
quantities similar to those produced endogenously in response to endotoxin,
causing piloerection and diarrhoea. Haemoconcentration, shock, metabolic
acidosis, transient hyperglycaemia followed by hypoglycaemia, and
hyperkalaemia were also observed. Gross and microscopic examination
demonstrated severe damage to target organs. Adrenal and pancreatic
haemorrhages were commonly observed. Polymorphonuclear leukocyte thrombi
were deposited in the major arteries of the lungs, and this was associated with
severe pneumonitis. Administration of low doses of cachectin (100 to 200 µg/kg
body wt. in rats) induces renal tubular necrosis, and ischaemic and haemorrhagic
lesions in the gastrointestinal tract (Beutler & Cerami, 1987).
To minimize inadvertent release of cachectin, its synthesis appears to be
tightly regulated as both transcriptional and post-transcriptional activation must
occur to allow its production (Beutler et al , 1986).
Glucocorticoids strongly antagonize the effects of endotoxin when
administered before the endotoxic insult. If glucocorticoids are added to
macrophage cultures cachectin synthesis will be completely inhibited. However,
glucocorticoids will only promote this action if added to the culture before the
macrophages are activated by endotoxin. Therefore, no inhibiting effect occurs if
dexamethasone is added after lipopolysaccharide induced-macrophage activation.
Cachectin appears to act by suppressing biosynthesis of several
adipocyte-specific mRNA molecules (Torti et al ,1985). Cachectin can also
induce the synthesis and/or release of specific proteins, including interleukin-1 by
monocytes and endothelial cells (Dinarello et al , 1986; Nawroth et al , 1986).
Cachectin can activate polymorphonuclear leucocytes stimulating their adhesion
to endothelial-cell surfaces and enhancing their phagocytic activity (Gamble et al
, 1985; Shalaby et al , 1985). Some other effects that may explain the action of
cachectin in vivo include stimulation of
the production of procoagulant activity, altering the properties of the vascular
endothelium and alteration of patterns of antigenic expresion (Beutler & Cerami,
1987).
Specific neutralization of cachectin and related cytokines may offer new
therapeutic tools to treat sepsis. It has been shown that mice treated with a
polyclonal antiserum directed against mouse cachectin become resistant to the
lethal effect of endotoxin (Beutler & Cerami, 1985). Therefore, it would seem
possible that neutralizing monoclonal antibodies directed against human cachectin
may prove to be useful in the treatment of sepsis, particularly in its early stages.
THE ROLE OF CORTICOSTEROIDS IN THE RESPONSE TO SEPSIS
The complex balance between the anti-inflammatory properties and the
physiological requirement for steroids may explain the conflicting conclusions
about steroid therapy in sepsis (Hess & Manson, 1983; Nohr & Meakins, 1985).
Although controversial, the clinical use of steroids in septic shock is common
practice and large doses of steroids (50-150 mg/kg body wt.) have been given
(Forse & Kinney, 1985). The rationale for the use of these agents in sepsis has
included the ability of these drugs to stabilize lysosomal and cell membranes, to
inhibit complement-induced granulocyte aggregation, to improve myocardial
performance (possibly due to inhibition of oxygen-free radical generation), to
modulate the release of endogenous opiates, to decrease arachidonate metabolites,
and to promote a shift in the oxyhaemoglobin dissociation curve to the right
(Karakusis, 1986).
Corticosteroids are important in maintaining normal physiology and their
concentration may or may not be increased in response to sepsis (Sibbald et al ,
1977). Elevation in steroid levels during sepsis may be a protective response to
decrease tissue damage by modulating inflammation. The effects of steroids on
specific immune processes are also complex. Leucocyte traffic is altered and
specific antibody responses are either not affected or enhanced by steroids (Nohr
& Meakins, 1985). In the liver, pharmacological doses of steroids have been
shown to diminish the disruptive effects of gram-negative bacteraemia and
endotoxin on the reticuloendothelial system (Balis et al , 1979). Steroids inhibit
margination and degranulation of polymorphonuclear leucocytes in the
microcirculation of the liver, lung and other organs, and may therefore be able to
inhibit complement activation and the release of factors which increase capillary
permeablity, the latter being implicated with the pathophysiology of multiple
organ failure syndrome (Duff, 1985).
Steroids block the synthesis of prostaglandins by inhibiting the release of
arachidonic acid from membrane phospholipids, and this may be one mechanism
by which steroids exert their beneficial effect in sepsis (Carlson et al , 1977;
Fletcher, 1982; Halla-Angeras et al , 1986). As arachidonate is the substrate of
both the cyclooxygenase and the lipoxygenase pathways, steroids also may inhibit
the synthesis of leukotrienes (Neeldeman et al , 1986; Samuelsson, 1983). Recent
evidence has shown a protective action of diethylcarbamazine, an inhibitor of
leukotriene biosynthesis, against the lethal effect of endotoxin in mice (Hagmann
et al , 1984). This supports the concept that leukotriene inhibition is a possible
alternative for the beneficial action of steroids during endotoxin and/or
infection.
Hall-Angeras et al (1986) have shown that the survival rate of rats
subjected to laparotomy and intravenous infusion of live E. coli , was
significantly improved by methylpredinisolone or diethylcarbamazine. However
this protective effect appeared not to be related to inhibition of prostaglandin
synthesis, but might have been due to inhibition of production of leukotrienes.
Glucocorticoids also act as important positive modulators of hepatic and
renal gluconeogenic enzymes, as they play a permissive role in the induction of
enzymes by other hormones, such as glucagon and epinephrine (Shackleford et al
, 1986). There is evidence that sepsis or endotoxin treatment are able to inhibit
gluconeogenesis (Filkins & Cornell, 1974; Guillem et al , 1982; Clemens et al ,
1984). Investigations into the cause of impaired gluconeogenesis have shown an
inhibition of the glucocorticoid induction of important gluconeogenic enzymes,
including
glucose-6-phosphatase,
tryptophan
oxygenase
and
phosphoenolpyruvate carboxykinase (Ripe and Berry, 1972; Guckian, 1973;
Cannonico et al , 1977; Shackleford et al , 1986). Therefore, interest has been
growing in the mechanisms of the endotoxin-glucocorticoid antagonism. Moore et
al (1976, 1985) have determined that inhibition is elicited by a protein mediator
released from macrophages, named "glucocorticoid-antagonizing factor" .
Glucocorticoids are known to act via intracellular receptors to which they bind
with high affinity after diffusion through the cell membrane (Higgins & Gehring,
1978).
These
steroid-receptor
complexes
suffer
conformational
change
("activation"), and then bind to acceptor sites in the nucleus, resulting in enhanced
transcription of a small percentage of genes (Schmidt & Litwack, 1982).
Shackleford et al
(1986), studying the hormonal regulation of
phosphoenolpyruvate
carboxykinase
in
endotoxin
treated
mice,
have
demonstrated no effect of endotoxaemia on binding, and on affinity or number of
glucocorticoid receptor binding sites, ruling out down-regulation of receptors as
the mechanism responsible for the inhibition of induction. They reached the
conclusion that endotoxin does not act at the early events in the glucocorticoid
induction process, but might act at a subsequent step.
Very recent evidence shows glucocorticoids are able to completely inhibit
cachectin production, a hormone released by endotoxin-activated macrophages,
provided they are applied to macrophage cultures in advance of activation by LPS
(Beutler & Cerami, 1987).
THE COUNTER-REGULATORY HORMONES AND THE REGULATION OF
THE METABOLIC RESPONSE TO SEPSIS
There are difficulties in understanding hormonal mechanisms involved in
producing the responses of the flow phase of injury or sepsis, and in fitting the
time-course of the action of a specific hormone, or group of hormones, to the
time-sequence of the metabolic changes (Frayn, 1986).
Because of their known general catabolic effects, the counter-regulatory
hormones, including adrenaline, cortisol, glucagon and growth hormone (Cryer,
1981), have received considerable attention in the study of sepsis. The action of
these hormones, in concert, can stimulate lipolysis, glycogenolysis and
gluconeogenesis (Frayn, 1986). Infusion of catecholamines with glucagon and
cortisol has been shown to mimic most of the metabolic features of the flow phase
after injury, such as increased metabolic rate with elevation of glucose turnover,
increased gluconeogenesis and negative nitrogen balance (Bessey et al , 1984;
Gelfand et al , 1984). However, contrary to the concept that counter-regulatory
hormones are the main regulators of the changes of the flow phase, is the fact that
neither the concentrations, nor the time course of changes in the concentrations of
these hormones in injured patients agree with those described in the infusion
studies (Frayn, 1986). Watters et al (1986) infusing hydrocortisone, glucagon and
epinephrine, with or without the inflammatory agent etiocholanolone, in healthy
volunteers, reached the conclusion that both inflammatory and endocrine
mediators are necessary to stimulate the responses to sepsis.
SEPSIS AND THE EFFECTS OF INSULIN
Insulin is perhaps the most studied, but the least understood homone in its
role during the acute response to sepsis. Many of the early features of the
response characterized by fuel mobilization, such as, depletion of glycogen
reserves and mobilization of triacylglycerol might be explained by a decrease of
insulin secretion (Frayn, 1986), possibly due to supression of insulin release by
the higher concentrations of catecholamines found in sepsis (Porte & Robertson,
1973; Biesel & Wannemacher, 1980). However, although plasma insulin
concentration has been reported to be decreased in experimental sepsis (Kelleher
et al , 1982; Lang et al , 1984) and in the septic man (Clowes et al , 1966), it has
also been shown to be increased during experimental sepsis (Neufeld et al ,1980
and 1982; Biesel & Wannemacher, 1980) and in the septic man (Long et al ,1985;
Shaw et al , 1985). Higher concentrations of insulin have also been reported
during endotoxin treatment (Yelich & Filkins, 1980; Neufeld et al , 1982). This
variability in insulin concentration may be explained by differences in the
experimental model of sepsis used, the changes in the type and source of sepsis,
nutritional status and the time-point at which the observations were made. Despite
this variability, the metabolic responses seem to be relatively uniform and insulin,
therefore, appears to act inefficiently during infection and is unable to produce its
anabolic effects.
Insulin Resistance
One unified way of looking at the endocrine control of the metabolic
changes after injury/sepsis is the unresponsiveness of the 'net storage' or the
anabolic processes to the elevation in insulin concentration. Therefore, glucose
production in vivo appears to be enhanced while glucose storage is reduced in the
presence of high insulin (Black et al , 1982). Despite insulin's to promotion of
normal fat
storage, net fat mobilization and oxidation are increased (Stoner, 1983; Frayn et
al , 1984).
The striking positive relationship between the time course of the increase
in plasma insulin with the amount of nitrogen excretion, further illustrates the
failure of normal insulin action in injured patients (Frayn, 1986). Perhaps, a
possible explanation to this finding is the fact that the same factors (interleukin1/proteolysis-inducing factor) may be involved in the stimulation of both insulin
secretion (George et al , 1977) and protein degradation (Clowes et al , 1983 and
1986).
Despite the fact that the general concept of insulin resistance during
injury/sepsis has been established for many years (Butterfield, 1955; Hinton et al ,
1971; Beisel & Wannemacher, 1980), there is a lack of understanding of the
action of insulin at the tissue level, from receptor through possible "signals" to
target pathways. Black et al (1982) using a modification of the euglycaemic
insulin clamp technique to study injured patients, suggested that the sensitivity of
the insulin receptor to circulating insulin is normal, and that insulin resistance in
peripheral tissues (probably skeletal muscle) may be caused by a postreceptor
defect. Clemmens et al (1984) studying isolated perfused livers showed a
resistance to the effects of insulin on gluconeogenesis in the livers of septic rats
(caecal-ligated and punctured rats), as compared to the livers of sham-operated
rats. Hasselgren et al (1987) reported some evidence for insulin resistance of
protein breakdown in septic muscle, while the response of amino acid transport
and protein synthesis to the hormone was not altered in sepsis.
BIOLOGICAL RESPONSE MODIFIERS DURING SEPSIS
All treatments, procedures and agents used to restore homeostasis and
maintain normal anatomy and physiology are regarded as biological response
modifiers (For review see: Meakins, 1981; Nohr & Meakins, 1985). Despite the
difficulties in selecting the appropriate modifier, and evaluating their action, they
appear to hold
some promise in modifying the metabolic response to
injury/sepsis. A list of some of the biological response modifiers is shown below
(Nohr & Meakins, 1985).
Biological agents
Endogenous
Tissues and cells
Hormones
Passive immunization
Target-directed monoclonal antibodies
Exogenous
Specific: active immunization
Non-specific: adjuvants
Bacillus Calmette-Guerin (BCG)
Muramyl dipeptide (MDP)
Zymosan
Drugs
Corticosteroids
Free radical scavengers
Iron-binding compounds
Prostaglandin related drugs
Levamisole
Opiate antagonists
Insulin release inhibitors
Others
General Methods
Restore anatomy and physiology
Surgery
Nutrition
Plasmapheresis
Fever
Some of these modifiers will be briefly considered. Thymic extracts,
namely a derivative of thymopoetin, TP5, increases survival in a septic burned
animal model (Waymak et al , 1984).
The role of some hormones (steroids and counterregulatory hormones) has
already been mentioned.
The important role of endotoxin (LPS) in the pathogenesis of sepsis
suggests that antibody to LPS may be therapeutically useful as an anti-toxin.
However, the most immunologically accessible portion of LPS (the O antigen
side chain) varies among gram-negative bacteria. Nevertheless, animal models
and a human trial using human anti-E. coli J5 antiserum have shown protection
against the lethal effects of gram-negative sepsis (Dunn & Ferguson, 1982;
Ziegler et al , 1982). It has also been demonstrated (Beutler et al , 1985) that mice
treated with a polyclonal antiserum directed against mouse cachectin become
resistant to the lethal effects of lipopolysaccahride (LPS). Thus the use of
monoclonal antibodies may also have potential therapeutic applicability.
Fibronectin is an opsonic glycoprotein important in promoting
phagocytosis by cells of the reticuloendothelial system. Plasma levels of this
glycoprotein
are decreased in septic patients.
Scovill et al (1978) have
shown that the administration of fibronectin as
cryoprecipitate can restore some aspects of host physiology towards normal.
Zymosan, a cell wall preparation of Saccharomyces cerevisiae , which can
attract and activate leucocytes enhancing their phagocytic properties (Williams et
al , 1982), has been used as a pre-treatment (injected intraperitoneally) in rodents
and successfully decreased mortality due to peritonitis (Joyce et al ,1978). On the
other hand, Goris et al
(1986) advocate the use of zymosan, also
intraperitoneally, to induce multiple organ failure and 'sepsis without bacteria'.
The role of prostaglandin inhibitors in the modification of the metabolic
response to sepsis has also being discussed previously.
Opiate receptors in the central nervous system may mediate some effects
of septic shock, possibly in conjunction with opiate receptors in the periphery
(Nohr & Meakins, 1985). Administration of the opiate antagonist, naloxone
hydrochloride, can reverse the hypotension of endotoxin shock to varying
degrees, to increase mean and pulse blood pressure, elevate respiratory rate,
prevent acidosis, attenuate hypoglycaemia, stabilize lysosomal membranes and
elevate white blood cell and platelet concentrations (Groeger et al , 1983;
Hinshaw et al , 1984; Ipp et al , 1984). However, some of the reasons to question
their therapeutic value for septic shock include studies evaluating survival time,
which have shown no long-term survival benefits; naloxone's cardiovascular
effects have mostly been assessed during the early phase of shock; the effects on
cardiovascular parameters due to naloxone are often short-lived and minimally
beneficial; despite increasing arterial pressure naloxone may also produce adverse
actions, such as regional vascular resistance. Species variability has been reported
for some of naloxone's vascular actions; and finally, naloxone stimulates the
sympathoadrenal system which may impair perfusion of the microcirculation
(Hinshaw et al , 1984).
Hyperinsulinaemia is a common event as part or the metabolic response to
sepsis (Biesel & Wannemacher, 1980). One view is that the metabolic changes of
sepsis is due to the lack of response to the anabolic action of insulin (Frayn ,
1986). Hyperinsulinaemia, itself, has been implicated as the cause, rather than the
product of insulin resistance (Marangou et al , 1986). Therefore, drugs
modulating insulin release can also be used not only to assess a possible reversal
in insulin sensitivity during sepsis, but also to try to reverse some of the metabolic
changes mediated under the conditions of elevated plasma insulin. Diazoxide
(Henquin et al , 1982), mannoheptulose (Simon et al , 1972) and streptozotocin
(Bolaffi et al , 1986) are examples of such drugs and their action during sepsis
will be discussed in later Chapters.
Fever
It has been observed clinically that the absence of hyperthermia during
sepsis may be associated with a poor outcome. Fever can increase leucocyte
capacity to mobilise, to kill and to react to bacterial antigens as well as promoting
the bacteriostatic effects of serum ( Kluger, 1981; Macrowiak & Marling-Casan,
1983; Nohr & Meakins, 1985).
Surgery
Although
surgery
itself
is
immuno-suppressive,
delayed
type
hypersensitivity responses can be improved by the drainage of visceral abscesses
(Meakins et al , 1979). In addition, the drainage of an intra-abdominal abscess can
also restore the function of 'failed' organs (Polk & Shields, 1977), so that organ
failure should be regarded as a potentially reversible 'organ dysfunction' (Bohnen
et al , 1983).
Nutrition
Refeeding via oral or intravenous route has been shown to be valuable in
correcting several abnormalities in immunological function. Total parenteral
nutrition is capable of restoring skin test reactivity, restoring lymphocyte
transformation, correcting serum immunoglobulin levels and restoring specific
antibody responses and
neutrophil chemotaxis (Nohr & Meakins, 1985). However, the choice of
the best method of nutritional support during sepsis, whether by the
use of enteral or parenteral route, is still controversial (Elwyn, 1980; Kettlewell,
1982). While a number of studies have claimed excess carbohydrate intake may
serve as a physiological stress rather than a nutritional support, others have
questioned the effect of intravenous fat in imparing the reticuloendothelial system
function (Jastrand et al , 1978; Carpentier et al , 1979; ). Despite controversial
results, amino acid solutions enriched with branched-chain amino acids have been
advocated in trying to correct the amino acid inbalance caused by sepsis (Freund
et al , 1978; Cerra et al , 1985; Harry et al , 1986). The infusion of carnitineindependent fats (short and medium chain fatty acids) as energy source appears to
be promising (Denninson et al , 1986). Nevertheless, the aim of nutritional
support in the hypermetabolic septic patient should be to provide appropriate
proportions of the substrates based on the observed metabolic alterations in order
to minimise complications and maximise benefit.
OBJECTIVE OF THE STUDY
Sepsis is an important clinical problem causing morbidity and mortality in
surgical patients (Cerra, 1982; McGowan & Gorbach, 1983; Wilmore et al , 1983;
Meakins, 1985). Survival depends on the adequacy of the immunological
reactions to contain and eliminate invading microbes, and on a series of integrated
physiological and metabolic responses necessary to maintain energy production
and cell function thoughout the body (Clowes et al , 1985).
The liver has a central role in intermediary metabolism during sepsis
(Wannemacher et al , 1979; Cerra et al , 1979), and yet liver function is
frequently disturbed by sepsis (Royle & Kettlewell, 1980; Duff, 1985).
It is well recognized that human clinical studies of sepsis are not easy to
perform, not only because of restrictions on their scope for ethical reasons, but
also because of the difficulty in establishing comparable groups of patients. On
the other hand, there are many experimental animal models of sepsis, each with
inherent characteristics, which are sometimes of questionable relevance to clinical
practice. It is therefore important to study the fundamental changes in liver
metabolism, induced by sepsis, in an experimental model which mimics as closely
as possible the clinical situation.
It is clear that the effect of infection varies with time and yet, surprisingly,
there is no information on the time-course of changes within a single experimental
model of sepsis.
We have therefore studied the temporal changes of the effects of
peritonitis on the hepatic metabolism of the rat. The rat liver behaves remarkably
like the liver.\\\ccv v00v.bv.b of the human, and the particular
septic model used is very reproducible and mimics abdominal sepsis in humans
(Wichterman et al , 1980; McGowan & Gorbach, 1983 ). The elucidation of the
metabolic changes within the liver and their relationship to hormonal levels could
have important clinical consequences. A comparison between the changes in
hepatic metabolism during sepsis, and those found in parenterally fed patients was
made in the last Chapter.
CHAPTER 2
MATERIALS AND METHODS
Animals
Sham-operated rats
Preparation of blood, plasma and tissue samples
Insulin determination
Measurements of lipogenesis and cholesterolegenesis
Preparation of isolated liver cells
Calculation of rates
Determination of enzyme activities in liver:
PEPCK
Glucose-6-phosphatase
HMG CoA Reductase
[1-14C]Oleate, [14C]HMGCoA, [3H]mevalonolactone and 3H2O were
purchased from the Amersham International, Amersham, Bucks, U.K.
Others
biochemicals and enzymes were obtained from Boehringer Corp. (London)Ltd., Lewes,
Sussex, U.K.
Animals
Male rats of the Wistar strain weighing about 250g were fed ad libitum on a
breeding diet for rats and mice (Special Diet Services, Witham, Essex, U.K.) containing
4% w/w lipid, 21% crude protein and 52% carbohydrate; the residue was non-digestible
material. The animals were maintained at an ambient temperature of 22±2° under a
12h light/12h dark cycle (lights on 07.30h).
Operations were carried out under light ether anaesthesia. A midline
laparotomy was performed. The caecum was mobilized by incising the mesocaecum
and the faeces were milked into the caecum, which was then ligated with a single 3-0
silk ligature in such a manner that bowel continuity was maintained. The antimesenteric surface of the caecum was punctured once with a 21-gauge needle, and
the bowel returned to the abdominal cavity. The abdominal wall was closed in two
layers and all rats received 0.9% (v/v) NaCl (2.5ml/100g body wt.) subcutaneously.
With this procedure an experimental peritonitis is created (Wichterman
et al, 1980) with a microbial flora that closely approximates that of human peritonitis
(McGowan & Gorbach, 1983) and therefore has proven to be a suitable model of the
human disease. Water was offered ad libitum.
The rectal temperatures of the septic rats were not different to those of the
sham-operated group. It has been reported that blood pressure of rats made septic by
this procedure is unchanged (Wichterman et al, 1980). The single puncture with a
small neddle gauge (21 G) was chosen so that the peritonitis could develop at a slower
rate allowing animals to starve and survive up to 48 h. The mortality rate was 3.7%
(one out of 27) at 12 h, 5.5% (two out of 36) at 24 h and 18.9% (15 out of 79) at 48 h ,
which was lower than that observed by Wichterman et al (1980) using a single
puncture operation.
Sham-operated rats
In the sham-operated animals the caecum was also mobilized by incising
the mesocaecum and then immediately returned to the abdominal cavity. All the other
steps of the operation and post-operative care were similar to those described for the
caecum-ligated animals. Sham-operated rats recovered more promptly from the
anaesthesia and did not show any of the signs of sepsis, as described by Wichterman
et al (1980), at the various times they were studied.
A group of animals was subjected to total interruption of their bile acid
hepato-enteric circulation. In these animals, after the midline laparotomy was
performed, all the small bowel was mobilized up to the duodenal-jejunal junction so
that the bile duct entry into the duodenum could be visualized. The bile duct was
isolated at approximately one centimeter from the entry point and ligated with a single
3-0 silk ligature . The caecum was then mobilized and all the other steps of the
operation and the post-operative care were the same as to those in
either
experimental (caecum-ligated and punctured) or control (sham-operated) animals.
Rats (starved 12, 24 or 48h post-operation) were anaesthetized with
pentobarbital (50mg/kg body wt.), injected intraperitoneally,
and a laparotomy
performed. Arterial blood from the abdominal aorta was collected using heparinized
syringes (2 ml) and the liver removed.
Part of the latter was immediately freeze-
clamped (Wollenberger et al, 1960). This was achieved by freezing the tissue between
aluminium tongs cooled in liquid nitrogen (i.e. at a temperature of about -190°C) which
lowered the temperature of the tissue to about -80°C in less than 0.1 sec. If not used
for metabolite determination on the same day, freeze-clamped livers were kept stored
in aluminium foil in a liquid nitrogen-refrigerator tank. To measure hepatic metabolites
freeze-clamped livers were ground to a fine powder with liquid nitrogen-cooled
porcelain mortar and pestel (Williamson et al, 1967) and extracted in ice-cooled
0.75M HClO4. This rapidly inactivated and precipitated protein
which was removed by centrifugation at 15000 rpm for 20 min. This HClO4 extract was
then neutralised with a 20% solution of potassium hydroxide (KOH) (Analar, BDH) and
the metabolic intermediates measured by spectrophotometric methods. The remaining
part of the liver was used for measurements of lipid and cholesterol synthesis or
determination of enzyme activity. Metabolites were extracted from total blood with
0.75M HCl04 as previously described by Williamson et al (1967). The tubes containing
blood and HClO4 were centrifuged at 3000 rpm for 10 min. The supernatant was
decanted into pre-weighed labelled 10ml plastic tubes which were then reweighed.
One drop of universal indicator (BDH) was added to the supernatant followed by a 20%
solution of potassium hydroxide (KOH) (Analar, BDH), which was added drop-wise until
the pH was between 7 and 8. After allowing to stand in ice for 10 min, the tubes were
reweighed and centrifuged at 3000 rpm for 10 min, and the supernatant, now a neutral
extract, was used for metabolite analysis. Pyruvate and acetoacetate were measured
immediately after neutralisation and the other metabolites were measured on the same
day of sampling or after storage at -20°C within two weeks. Part of the total blood
sample was centrifuged at 3000 rpm for 10 min and the plasma was separated and
used for determination of free fatty acids, triacylglycerol and insulin concentrations and
measurement of the specific radioactivity of plasma water.
Before giving details about the various enzymatic methods used to determine
metabolite concentrations, the
principles behind enzymatic analysis are introduced.
The enzymatic assay of metabolic substrates is based on the principle that a
specific enzymatic reaction in which the substrate participates is coupled with the
reduction of NAD/NADP or oxidation of NADH/NADPH. The pyridine nucleotides (NAD,
NADP) absorb light at 260 nm, and in the reduced state (NADH, NADPH) they have an
additional absorption band with a maximun at 340 nm. By measuring the optical density
at 340 nm, the enzymatic conversion of the substrate can be followed directly in a
spectrophotometer cuvette. Regardless of whether NAD accepts H+ or whether NADH
donates H+, at this wavelength the optical density increases or decreases by 6.22 units
(light path 1 cm) with the production or consumption of 1 µmole of NADH/NADPH.
Since in a specific enzymatic reaction, 1 µmole of substrate usually co-reacts with 1
µmole of NAD/NADP (or NADH/NADPH), the change in optical density will reflect
accurately the amount of substrate consumed by the reaction. Provided the assay
conditions are optimum, conversion of the substrate is practically complete and the
optical density difference (ODD) can be used to calculate the concentration of the
substrate in the blood or tissue sample by multiplying with an appropriate dilution
factor.
Since many enzymatic reactions are equilibrium reactions, in order to make
an end-point measurement the equilibrium of the reaction has to be displaced such that
it favours the complete consumption of the substrate. The reaction equilibrium can be
influenced by several factors such as increase in substrate or cofactor concentration,
variation of pH, presence of trapping agents, or the use of regenerating reactions in
which one of the co-substrates may be regenerated by a secondary reaction. If none of
the
reactants
or
products
of
an
enzymic
reactions
is
measurable
spectrophotometrically, it is often possible to transform one of
the products by another enzymatic reaction which in turn can be easily measured (e.g.,
measurement of glucose, non-esterified fatty acids and triacylglycerols). The former
reaction, in which the substrate to be determined is transformed, is known as the
auxiliary reaction, whereas the reaction used for the actual measurement is known as
the indicator reaction. Both reactions can usually be carried out in the same assay
mixture (Bergmeyer H.-U.,1963).
Specificity of an enzymatic assay depends on the purity of the enzyme
preparation whereas precision depends on the provision of optimum assay conditions.
The sensitivity of enzymatic assays is limited by the fact that sufficient conversion of
NAD/NADP (or NADH/NADPH) must occur to the extent of producing a measurable
change in the optical density.
The following metabolites were measured by standard enzymatic methods
as described below:
Determination of D-Glucose
Blood and liver glucose were measured according to the method described
by
Slein (1963).
Reaction sequence:
a. Auxiliary reaction
Mg++
GLUCOSE + ATP
——————————————>
GLUCOSE-6-
PHOSPHATE + ADP
b. Indicator reaction:
Mg++
glucose-6-phosphate
GLUCOSE-6-PHOSPHATE
dehydrogenase
——————————————————————>
6-
PHOSPHOGLUCONATE
+ NADP+
+ NADPH +
H+
At pH 7.5, the equilibrium for the indicator reaction is far to the right which
ensures the completion of both reactions (since glucose-6-phosphate formed
in the former is rapidly used up in the latter reaction). Although hexokinase
catalyses the phosphorylation of several other monosaccharides, specificity
is
provided by glucose-6-phosphate dehydrogenase (G6PD) with which hexose
or pentose esters other than glucose-6-phosphate do not react.
Buffer solution for assay:
20ml 0.1M tris buffer pH 8.0
2ml 0.1M magnesium chloride
2ml 0.01 M ATP
2ml 1% NADP
0.13ml of G6PD (1mg/ml)
This was prepared freshly for each assay. The total volume in each cuvette
was 2.0ml. In the sample cuvettes, this consisted of 0.1ml of neutralised
HClO4 extract, 0.9ml of distilled water and 1ml of assay buffer; in the
control cuvette 1ml of water and 1ml of assay buffer was added. The
cuvettes were read at 340nm before , at 10min and 15min after addition of 0.010ml
of hexokinase.
Measurement of L-(+)-Lactate
Concentrations of blood and liver lactate were measured according to the
method described by Hohorst (1963).
Reaction sequence:
Lactate dehydrogenase
LACTATE + NAD+ ——————————————> PYRUVATE +
NADH + H+
The equilibrium of this reaction lies well on the side of lactate and NAD.
Therefore, in order to ensure the complete conversion of lactate, the reaction
products have to be removed from the equilibrium. Protons are trapped by an
alkaline reaction medium; the pyruvate reacts with hydrazine hydrate in the
buffer solution to form pyruvate hydrazone and, in addition, a large excess of
NAD and enzyme is used to obtain a sufficiently rapid end point. Lactate
dehydrogenase reacts only with L-(+)-lactate and thus provides specificity
for the assay.
Buffer solution for the assay:
40ml 0.2 M Tris
5ml hydrazine hydrate 100%
25mg EDTA
Made up to 100ml of distilled water.
The pH of the buffer solution was adjusted to pH 9.5 with 5M hydrochloric
acid and it was stored for up to two weeks at 4°C. Before use, 1 ml of 1%
(w/v) NAD was added to every 10 ml of assay buffer. The total volume in
each cuvette was 2.0ml. In the sample cuvettes this consisted of 0.2ml of
neutrilised HClO4 extract, 0.8ml of water and 1ml of assay buffer; the
control cuvette contained 1ml of water and 1ml of assay buffer. All cuvettes
were read at 340 nm before and at 35 and 45 min after the addition
of
0.02ml of lactate dehydrogenase.
Measurement of Pyruvate and Acetoacetate
Pyruvate and acetoacetate share very similar assay conditions and therefore
were measured sequentially in the same sample (same cuvette) according to
combination of methods respectively described by Hohorst et al (1959) and
a
Williamson et al (1962).
Reaction sequence:
lactate dehydrogenase
PYRUVATE + NADH + H+
————————————————————>
LACTATE + NAD
ACETOACETATE
+
3-OH-butyrate dehydrogenase
NADH
+
H+
—————————————>
3-
HYDROXYBUTYRATE + NAD+
The equilibrium of the first reaction at pH 7.0 is sufficiently far to the right to
ensure a quantitative measurement of pyruvate levels provided that the
NADH
concentration is not less than 0.01 mM. At the same pH and with a
suitable
excess of NADH, at least 98% of the acetoacetate is reduced to
D-(-)-3-
hydroxybutyrate. However, due to the low activity of
3-hydroxybutyrate
dehydrogenase preparations the second reaction proceeds
at a much slower
rate than the former.
10ml of 0.1 Potassium phosphate buffer pH 6.9
1 ml of 0.5% (w/v) NADH
cuvette
A fresh solution was prepared for each assay. The total volume in each
was 2 ml. In the sample cuvettes, this consisted of 1ml of neutralised HClO4
extract and 1ml of assay buffer. The control cuvette contained 1ml of water
and 1ml of assay buffer solution. The cuvettes were read at 340 nm before
and
10min after the addition of of 0.01ml of lactate dehydrogenase.
Hydroxybutyrate dehydrogenase (0.01ml) was then added to
they were read again at 35 and 45 min thereafter.
3-
each cuvette and
Measurement of D-(-)-3-hydroxybutyrate
Blood
and hepatic concentrations of 3-hydroxybutyrate were measured
according to the method described by Williamson et al (1962).
Reaction sequence:
3-hydroxybutyrate dehydrogenase
3-OH-BUTYRATE + NAD+ ————————————>
NADH + H+
ACETOACETATE +
The equilibrium of the reaction at pH 8.0 is reached when approximately
40% of the 3-hydroxybutyrate is oxidised to acetoacetate. However, the
presence of hydrazine in the buffer solution traps the acetoacetate formed as
a
hydrazone and the reaction proceeds quantitatively from the left to the right.
70 ml 0.1M tris buffer pH 8.5
0.25 ml of hydrazine hydrate 100%
25mg of EDTA
Made up to 100 ml with distilled water.
The pH of the assay buffer solution was adjusted to pH 8.5 with 5M
hydrochloric acid and it was stored for up to two weeks at 4°C. Before use, 1
ml of 1% (w/v) NAD was added to 10ml of assay buffer for each assay. The
total volume in each cuvette was 2 ml. In the sample cuvettes, this consisted
of 0.5 ml of neutralised HClO4 extract, 0.5ml of water and 1 ml of assay
buffer; the control cuvette contained 1 ml of water and 1 ml of assay buffer.
The cuvettes were read at 340 nm before and 50 and 60 min after the
addition of 0.01 ml of 3-hydroxybutyrate dehydrogenase.
Measurement of L-Alanine
Blood and hepatic concentrations were measured according to the method
described by Williamson et al (1967).
Reaction sequence:
alanine dehydrogenase
ALANINE + H2O + NAD+ ——————————————> PYRUVATE +
NH4+ + NADH
In the presence of low H+ ion concentration, at pH 9.0, this reaction
proceeds
quantitatively from the left to the right. Hydrazine hydrate is included in the
buffer solution in order to trap the pyruvate formed by conversion to
pyruvate hydrazone.
40ml 0.2M tris buffer
25mg EDTA
The pH of the buffer solution was adjusted to pH 9.0 with 5 M hydrochloric
acid and it was stored for up to 14 days at 4°C. Before use, 1ml of 1% (w/v)
NAD was added to 10ml of assay buffer for each assay. The total volume per
cuvette was 2 ml. In the sample cuvettes, this consisted of 0.5 ml of
neutralised HClO4 extract,0.5 ml of water and 1 ml of assay buffer; the
control cuvette contained 1 ml of water and 1 ml of asssay buffer.All cuvettes
were read at 340 nm before and at 50 and 60 min after addition of
0.01ml of alanine dehydrogenase.
Determination of D-3-Phosphoglycerate, D-2-Phosphoglycerate
and
Phosphoenolpyruvate and Pyruvate
Hepatic concentrations of pyruvate, PEP, 2-phosphglycerate and
phosphoglycerate were measured according to the method described by Czok
3&
Eckert (1963).
Reaction sequence:
a.
LACTATE + NAD+
——————————————> PYRUVATE +
NADH + H+
pyruvate kinase
b.
PYRUVATE
+
PHOSPHOENOLPYRUVATE + ADP
Mg2+, K+
ATP
———————————>
enolase
c.
PHOSPHENOLPYRUVATE
———————————>
2-
PHOSPHOGLYCERATE
Mg2+
phosphoglycerate mutase
d.
2-PHOSPHOGLYCERATE
—————————————>
3-
PHOSPHOGLYCERATE
At pH 7.4 quantitative conversion of the over-all reaction is assured because
of the positions of the equilibrium state of the reactions catalysed by pyruvate
kinase and lactate dehydrogenase being far to the right. 3-Phosphglycerate
can only be determined under the conditions described here if the assay
mixture contains less than 10-3 M inorganic phosphate. The determination of
2-phosphoglycerate, phosphoenolpyruvate and pyruvate is not affected by
phosphate. Specificity is secured by the high specificity of the reactions
catalysed by enolase and pyruvate kinase.
10 ml Tris HCL buffer ph 7.4 0.1M
1 ml Mg Cl2 0.1 M
3 ml
KCl 1.0 M
1 ml
ADP 0.02 M
0.5 ml
NADH 0.5 %
This was prepared freshly for each assay. The total volume in each cuvette
was 2.0 ml. In the sample cuvettes, this consisted of 0.5 ml of neutralised
liver HClO4 extract, 0.5 ml of distilled water and 1 ml of assay buffer; in the
control cuvette 1 ml of water and 1 ml of assay buffer were added. The
cuvettes were read at 340 nm before and for 5-10 min after the addition of
lactate dehydrogenase (0.01ml); pyruvate kinase (0.01 ml) was added and
the changes in the optical density followed for 5-10min; enolase (0.01ml)
was added and the cuvettes were read continuously for 5-10 min; finally
phosphoglycerate mutase (0.01 ml) was added to the cuvettes and the
change
in the optical density followed for 15-20 min. All cuvettes were read using a
Gilford recording spectrophotometer.
Measurement of Dihydroxyacetone Phosphate,
Glyceraldehyde-3-phosphate and Fructose-1,6-bisphosphate
D-
Hepatic concentrations of dihydroxyacetone phosphate (DHAP),
glyceraldehyde-3-phosphate (GAP)
and fructose-1,6-bisphosphate (FDP)
were determined according to the method described by Bücher & Hohorst
(1963).
Reaction sequence:
GDH
a.
L-(-)GLYCEROL-1-PHOSPHATE + NAD+ —————> DHAP +NADH
+ H+
TIM
b.
DIHYDROXYACETONE PHOSPHATE ——> GLYCERALDEHYDE -3-
PHOSPHATE (GAP)
aldolase
c.
GAP + DHAP ———————> FRUCTOSE-1,6-BISPHOSPHATE
(FDP)
The equilibrium state of the reactions ensures that the overall
reaction
proceeds quantitatively. For each mole of FDP formed 2 moles of NADH are
oxidised, therefore, the inclusion of the triosephosphate isomerase (TIM)
reaction increases the sensitivity of the assay.
10 ml
Tris HCl ph 7.4
1 ml
NADH 0.5%
The total volume in each cuvette was 2 ml. In the sample cuvettes, this
consisted of 1 ml of neutralised liver HClO4 extract and 1 ml of assay buffer
solution; the control cuvette contained 1 ml of distilled water and 1 ml of
assay buffer. All cuvettes were read continuously using a Gilford
spectrophotometer as follows: before and for 5-10 min after addition of
glycerol-3-phosphate dehydrogenase (GDH); before and for 5-10 min after
addition of triophosphate isomerase (TIM) and before and for 10-15 min
after addition of aldolase.
Measurement of Adenosine-5'-Triphosphate and
D-glucose-6-
phosphate
Adenosine-5'-triphosphate (ATP) and glucose -6-phosphate were measured
with a combination of the methods described by Lamprecht &
(1963) and Hohorst (1963); using the same cuvette.
Trautschold
Hexokinase
phosphorylates glucose with ATP in the presence of Mg++ to give glucose-6phosphate. Glucose-6-phosphate dehydrogenase catalyses the
oxidation
of
glucose-6-phosphate with NADP. Each mole of ATP forms 1 mole of NADPH.
Reaction sequence:
hexokinase
GLUCOSE + ATP ——————————> GLUCOSE-6-PHOSPHATE (G-6-P) +
ADP
G6P-DH
G-6-P
+
NADP+
———————> 6-PHOSPHOGLUCONO-LACTONE +
NADPH + H+
In the presence of equivalent concentrations of glucose and Mg2+, ATP is
virtually quantitatively converted to ADP by hexokinase. The equilibrium of
the glucose-6-phosphate reaction lies to the right.
10 ml
Tris
0.1M
pH 7.4
1 mg/ml NADP
2 ml
Mg Cl2 0.1 M
0.5 ml
2
Glucose
0.1 M
This was prepared freshly for each assay. The final volume per cuvette was
ml. In the sample cuvettes this consisted of 0.5 ml of liver HClO4 extract,
0.5 ml of distilled water and 1 ml of buffer solution. The control cuvette
contained 1 ml of distilled water and 1 ml of buffer solution.Cuvettes were
read at 340 nm before and 5-10 min after 0.005 ml of glucose-6-phosphate
dehydrogenase was added and subsequently 10-15 min after hexokinase
(0.005 ml) was added.
Determination of Acetyl-Coenzyme A
Hepatic Acetyl-CoA was measured by the method described by Decker
(1963).
Reaction sequence:
citrate synthase
ACETYL-SCoA + OXALOACETATE + H2O ———————> CITRATE + CoASH +
H+
malate dehydrogenase
L-MALATE + NAD+ ————————————————> OXALOACETATE +
NADH + H+
At pH 8.0 the equilibrium of the over-all reaction lies to the right and
acetyl-CoA is used quantitatively.
Buffer solution used for assay:
15 ml Tris 0.1 M
1 ml
L-malate
0.1 M
0.05 ml NADH
1 ml NAD
1 ml
ml.
pH 8.0
0.5 %
1%
This was made freshly for each assay. The total volume per cuvette was 2
In the sample cuvettes this consisted of 1 ml of liver HClO4 extract and 1 ml
of buffer solution. The control cuvette contained 1 ml of distilled water and 1
ml of buffer solution. Cuvettes were read before and at 15 min after addition
of citrate synthase (0.01 ml).
Determination of L-Glutamate and Glutamine
Hepatic glutamate was measured according to the method described by
Bernt &
Bergmeyer (1963). The determination of glutamine was also based on this
enzymatic technique.
Reaction sequence:
glutamate dehydrogenase
L-GLUTAMATE + NAD+————————————————> OXOGLUTARATE +
NADH + NH4+
glutaminase
L-GLUTAMINE –––––––––––––––––––––––––> L-GLUTAMINE + NH4+
The equilibrium lies far to the left. However, under alkaline conditions
9.0) and the presence of excess NAD, oxoglutarate can be trapped with
(pH
hydrazine
and glutamate quantitatively oxidized to oxoglutarate.
2 ml Hydrazine hydrate 100 %
20 ml Tris 0.2 M
25 mg EDTA
Made up to 40 ml with distilled water and the pH (9.0) adjusted using 5 N
HCl. This could be kept at 4°C for up to 14 days. To be used for assay
(cocktail), NAD (1mg/ml) and ADP (0.5mg/ml) were added to the buffer
solution. The total volume per cuvette was 2 ml. In the sample cuvettes this
consisted of 1 ml of liver HClO4 extract and 1 ml of cocktail. The control
cuvette contained 1 ml of distilled water and 1 ml of cocktail. Cuvettes were
read before and 30-40 min after the addition of glutamate dehydrogenase (in
glycerol).
Glutamine
In order to measure glutamine liver HClO4 extracts were pre-incubated in
the presence of glutaminase under the contitions described below:
0.5 ml
Liver extract
0.5 M Acetate buffer 0.5 M
0.01 ml NH2OH
1M
pH 4.8
0.02 ml Glutaminase [5 mg/ml]
The total volume per tube was made up to 1 ml with distilled water. An
internal standard was added, consisting of 1 mM glutamine (0.2ml) and
distilled water (0.8 ml). Tubes were mixed and incubated at 37°C for 45 min.
These were then cooled, 0.5 ml of the mixture taken and the concentration of
glutamate measured by the enzymatic method described above (Bernt &
Bergmeyer, 1963). As glutamine is converted to glutamate by glutaminase,
the
total
concentration
of
glutamine+glutamate
is
measured
spectrophotometrically. Therefore, the actual concentration of glutamine is
calculated by subtracting the concentration of glutamate measured in the
liver extract from the total concentration (glutamine + glutamate) found in
the glutaminase pre-incubated extract.
Measurement of Non-esterified fatty acids
The method of Shimizu et al (1979) was used for the measurement of
non-esterified fatty acids (NEFA), which is based on the activation of NEFA
by
acyl-CoA synthetase.
Reaction sequence:
acyl-Co A synthetase
NEFA + COENZYME A + ATP —————————> ACYL COENZYME A +
AMP+ PPi
myokinase
AMP + ATP ———————> 2 ADP
pyruvate kinase
2 ADP + PHOSPHOENOLPYRUVATE
————————->
2 ATP + 2
PYRUVATE
2 PYRUVATE + 2 NADH
LACTATE + 2 NAD+
————————————————> 2
The reaction calalysed by acyl-CoA synthetase favours the production of acyl
coenzyme A provided the pH is 8.0 and there are sufficient amounts of ATP
and coenzyme A. Therefore, the production of AMP is used as a measure of
NEFA activation through an indicator system provided by successive
reactions catalysed by myokinase, pyruvate kinase and lactate dehydrogenase. The
equilibrium state of all three reactions in this indicator system is sufficiently
far to the right to ensure that the overall rate of the reaction is
limited only by the
concentration of NEFA. Specificity for the method is
enhanced
fact that Acyl CoA synthetase only activates monocarboxylic
acids with 6 to 18
carbon atoms and does not affect other carboxylic acids and
lipids.
200 ml 0.1 M tris pH 8.0
35.1 mg EDTA (o.6 mM)
by
the
406.6 mg magnesium chloride (10mM)
2 ml Triton X-100
This was stored at 4°C. Immediately before each assay, the following
reagents were added to 36 ml of buffer solution:
1.6 ml NADH 0.5% (w/v)
1 ml
ATP 0.1 M
1 ml phosphoenolpyruvate 0.2 M
0.2 ml myokinase 2mg/ml
0.4 ml pyruvate kinase 1mg/ml
0.2 ml lactate dehydrogenase 10mg/ml
The total volume per cuvette was 2.005 ml. The sample cuvettes contained
0.04ml of plasma, 1.95ml of buffer mixture and 0.015ml of acyl-CoA
synthetase. The plasma volume was replaced by 0.04ml of water in the
control cuvette. All cuvettes were read at 340 nm before and 20 min after the
addition of coenzyme A. Further readings were taken at 5 min intervals until
the readings of the optical density were constant.
Measurement of Triacylglycerol
Plasma triacylglycerol concentration was measured according to the method
described by Eggstein & Kreutz (1966). Plasma triacylglycerol were
initially hydrolysed to glycerol and non-esterified fatty acids. The glycerol
was
measured
by
the
enzymatic
techique
described
below.
The
concentration of
measured in neutralised HClO4 extract was subtracted from the
free
total
glycerol,
glycerol
measured after alkaline hydrolysis of the plasma sample to
obtain
glyceride-glycerol concentration which, in turn, was taken to be
an estimate of the
the
triacylglycerol content of the plasma.
Reaction sequence:
a. Hydrolysis:
at 70°C
TRIACYLGLYCEROL + 3 H2O ————————> GLYCEROL + 3
FATTY ACIDS
alchoholic KOH
glycerokinase
pyruvate kinase
GLYCEROL + PHOSPHOENOLPYRUVATE ——————> GLYCEROL-3-PO4 +
LACTATE
ATP
ADP
ATP
NADH
NAD+
Hydrolysis:
To 0.2 ml of plasma 0.5ml of alcoholic KOH (0.5N KOH in 98% ethanol) was
added. The mixture was incubated at 70°C for 30 min and then cooled.
Thereafter, 1.5ml of 0.1 M MgSO4 was added to precipitate protein, and the
samples were centrifuged at 3000 rpm for 10 min. The triacylglycerol
present in plasma produced equimolar quantities of glycerol. This glycerol
produced by hydrolysis was contained in the supernatant solution, 0.5ml of
which was used for the enzymatic measurement of total glycerol.
Measurement of glycerol
Reaction sequence:
glycerokinase
GLYCEROL + ATP ———————————> GLYCEROL-3-PO4 +
ADP
pyruvate kinase
ADP
+
PHOSPHOENOLPYRUVATE
—————————>
ATP +
PYRUVATE
PYRUVATE + NADH + H+ ————————————> LACTATE +
NAD+
The equilibrium state for all three reactions can be acheived at pH 7.4 and in
the presence of Mg++ ions. It is far to the right to ensure that there is a
quantitative comsumption of glycerol in the first reaction and that the
indicator reactions can rapidly proceed to completion.
30 ml 0.1 M tris buffer pH 7.4
3 ml 0.1 M magnesium chloride
35 mg phosphenolpyruvate
50 mg ATP
12.5 mg
NADH
0.2 ml lactate dehydrogenase
0.2 ml pyruvate kinase
This was made up immediately before use and the pH adjusted to pH 7.4
with
0.2 M tris buffer.
Triacylglycerol
The total final volume per cuvette was 2 ml. In the sample cuvettes, 0.5ml of
the hydrolysed plasma sample was added to 1 ml of assay buffer and 0.5 ml
of
distilled water.
Glycerol
The sample cuvettes contained 1 ml of assay buffer and 1 ml of neutralised
HClO4 extract. Blank cuvettes contained 0.5 ml of buffer mixture and 1.5 ml
of distilled water. The control cuvette contained 1 ml of distilled water and 1
ml of assay buffer. All cuvettes were read at 340 nm before and 40 min after
addition of 0.01 ml of glycerokinase. They were then re-read every 10 min
until there was no further change in the optical density.
Calculations for blood and liver metabolites:
All metabolite calculations were based on the change in optical density
measured at 340 nm in the sample cuvettes following addition of enzyme, and after
subtraction of the non-specific change which occured in the control cuvettes.
Therefore:
Optical density difference (ODD) = Change in absorbance for sample cuvette - Change in
absorbance for control cuvettes
Since the molar extinction coefficient of NADH is 6.22 cm2/µmol, the amount of substrate in the
cuvette = ODD / 6.22 X total volume in cuvette.
This is multipied by the dilution factor for each sample to give the concentration of substrate;
In the blood:
In the liver: 1 g frozen liver = 0.8 ml ; 1 g liver + 4 ml PCA = 4.8
Insulin Determination
Insulin was measured by radioimmunoassay with a rat insulin standard
(Albano et al, 1972) using activated charcoal for separation of the free and bound
hormone. A disequilibrium assay was set up with a 3-day incubation period and all
plasma samples were assayed in duplicate with 20 µl of plasma in each tube. The
essential principle of the insulin asay, as for any other radioimmunoassay technique, is
the reaction of a fixed amount of specific insulin antibody with a mixture of the plasma
sample to be assayed and a constant amount of radioactively labbelled pure insulin.
The reaction is initiated by incubating the plasma sample with insulin antibody for
approximately 48 hours. The radioactively labelled insulin is then added to compete for
the remaining binding sites on the antibody complex. The reaction is allowed to
approach completion over the subsequent 24 hours, and the antibody-bound insulin is
separated from the free hormone by differential adsorption on to albumin-coated
charcoal. The distribution of the radioactivity between the free and bound forms of
insulin is then determined. It is expected that the amount of radioactively labelled
insulin bound to antibody to decrease as
the concentration of the unlabelled insulin in the plasma sample increases due to
competition for the limited number of binding sites available. Therefore, results
obtained with samples of unknown insulin concentration can be compared with
standard curves obtained by the measurement of samples to which known amounts of
pure insulin standard have been added.
Lipogenesis and cholesterolegenesis were measured in vivo with 3H2O as
previously described ( Jungas et al, 1968; Robinson et al, 1978). The rats were
injected intraperitoneally with 5 mCi (0.25ml) of 3H2O, and tissues removed one hour
later; 5 min before removal of tissue the rats were anaesthetized with pentobarbital
(50mg/kg body wt.). Heparinised blood was collected for measurement of the specific
activity of plasma water. The tissues (in duplicate into 50 ml tubes) were hydrolysed
under alkaline conditions by addition of 3 ml of 30% KOH and heated in a water bath at
70°C for 15 min. Absolute alcohol was then added and the samples returned to the
70°C water bath for a further 175 min (Stansbie et al, 1976). The non-saponifiable
lipids were extracted three times with light petroleum (b.p. 40-60°C; total vol. 20 ml)
and the organic extract was washed twice with distilled water and transfered to
scintillation vials. After evaporation of the solvent, the 3H radioactivity of the residue
was measured by scintillation counting in half of the samples to determine the total
incorporation of 3H2O into the non-saponifiable fraction. Cholesterol and squalene
were isolated from the non-saponifiable residue, from the other half of the samples, by
chromatography
on
thin-layer
plates
of
silica
gel
H
developed
with
chloroform/methanol (99:1, v/v). The plate was dried and the cholesterol-containing
band was located by spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v).
This area of the plate was scraped into a scintillation vial, and the 3H content of the
sterol fractions were determined by scintillation counting (Gibbons et al, 1983).
The aqueous phase after extraction of the non-saponifiable lipid was
acidified to pH 1.0 with 6N H2SO4, the labelled fatty acid fraction was extracted three
times with light petroleum and the organic extract washed twice with distilled water.
The extract was transferred into vials to dry (Stansbie et al, 1976) . After evaporation
of the solvent, the 3H radioactivity of the residue was determined by scintillation
counting.
Preparation of isolated liver cells
Hepatocytes were prepared essentially as described by Berry & Friend
(1969) with the modifications described by Krebs et al (1974). Routine tests indicated
that about 90% of the hepatocytes excluded Trypan Blue.
Operative technique, liver perfusion and isolation of hepatocytes
The rat was anaesthetized by an intraperitoneal injection of pentobarbital (50
mg/kg body wt.). Heparin (150 units; 0.15 ml) was then administered via a saphenous
vein. A laparotomy through a vertical mid-line incision was performed. Two midtransverse incisions were made to the left and the right of the mid-line to allow an
extended exposure of the abdominal cavity. The portal vein and the descending vena
cava were exposed by moving the intestines to the left side of the abdominal cavity.
One loose silk ligature
(3-0) was placed around the descending vena cava immediately above the right renal
vein, and two ligatures,
about 1 cm apart, were placed around the portal vein. The
portal vein was then cannulated using a 17 gauge metalic intravenous cannula. After
withdrawal of the inner needle, the cannula was secured by tying the ligature that was
closest to the liver. The other tie was tightened to prevent blood from flowing out of the
lower part of the portal vein and to tie off the common bile duct. The cannula was
therefore firmly in place and the appearance of blood back-flow at the cannula end
from the penetrated portal vein confirmed the success of the cannulation.
The thorax was immediately opened, through a transverse incision. A loose
ligature was placed around the inferior vena cava just below the heart. A 16 gauge
plastic cannula was used to cannulate this vessel through the right atrium and secured
with one 3-0 silk tie.
Liver perfusion and treatment with collagenase:
The method of liver perfusion is that described by Hems et al (1966). A
water-jacketed oxygenator was used and the temperature kept approximately at 38°C.
The perfusion medium used was the saline of Krebs & Henseleit (1932) from which
calcium is omitted because it decreases the activity of the enzyme. The total volume of
the perfusion fluid was initially 150 ml. The first 40 ml of the medium passing through
the liver were discarded in order to remove the bulk of the blood cells. At this point 30
mg of the crude collagenase (Boehringer Corporation, London), suspended in a small
volume of perfusion medium were added. Pure collagenase is not suitable because the
contaminating proteinases in the crude preparation play a very important role. The flow
rate was approximately 25 ml /min. The perfusion medium was kept in equilibrium with
O2/CO2 (95:5 ; v/v). The perfusion was continued for 20-30 min and any leaked
perfusion fluid appearing in the abdominal cavity was collected and returned to the
reservoir. The action of the
enzyme was reflected by the degree of leaking; when this had reached a rate of about
10 ml/min, usually after 25 min, the perfusion was discontinued and the liver was
transferred to a beaker. The medium used in the perfusion was added to
the broken-up liver tissue as this was put into a nylon cloth (mesh size 0.44 X 0.44 mm)
which was on the top of a 100 ml disposable plastic beaker. The liver tissue was then
gently broken apart with a pair of scisors and the cells teased out with a plastic
spatula, always keeping the perfusion medium (which already contained cells washed
out from the liver during the perfusion) flowing through the nylon cloth. The filtrate was
centrifuged at 50 g for 2 min and the cells were re-suspended in about 30 ml of the
Ca++-containing Krebs-Henseleit medium. The suspension was centrifuged again at
50 g for 2 min and the pellet weighed and resuspended in 16 volumes of the KrebsHenseleit medium containing Ca++, when it was ready to be incubated. Isolated
hepatocytes were used immediately after preparation.
The isolated hepatocytes were incubated with constant shaking, at 65
oscillations per min, for 20, 40 or 60 minutes in Krebs-Henseleit saline (Krebs &
Henseleit, 1932) containing essentially fatty acid-free albumin (final concentration 2.5%
w/v), the gas phase was O2/CO2 (95:5) and the temperature was 37.5° C. The total
incubation volume containing 65-95mg wet wt. of cells and added substrates (5mM
lactate, 5mM pyruvate, 5mM alanine, 5mM glutamine, 5mM dihydroxyacetone, 5mM
butyrate and 2mM [1-14C]oleate; 40µci/ml) was 4 ml. Incubations were stopped by
addition of 0.4ml of 20% (w/v) HClO4 , the mixture was centrifuged to remove
denatured protein and the supernatant was neutralized with KOH. The precipitate of
KClO4 was removed by centrifugation.
All incubations with [1-14C]Oleate were performed in duplicate in KrebsHenseleit saline (Krebs & Henseleit, 1932) containing dialysed albumin (fatty acid poor,
final concentration 2.5%, w/v). One of each pair was incubated in a 25ml Erlenmeyer
flask with a centre well and rubber seal. The incubation was stopped by the injection of
0.4 ml of 20 % (w/v) HClO4 with a long-needled syringe (Whitelaw & Williamson,
1977). Sodium hydroxide (NaOH; 0.3 ml) was injected into the centre well and
metabolic CO2 collected by shaking the flasks for a further 2 h at room temperature.
The NaOH was then removed for measurements of radioactivity and the deproteinised
incubation medium was treated as described above.
The reaction in the duplicate flask was stopped by centrifugation for 1 min at
5000 g to separate cells from the medium. Chloroform/methanol [2:1 (v/v); 20 vol.] was
added to the cell pellet and the lipids were extracted as described by Folch et al
(1957). The radioactivity in the chloroform phase was used as a measure of the [114C]Oleate converted into esterified fats. The disappearance of radioactivity which was
extracted by chloroform from the incubation medium (minus pellet) was used to
calculate the oleate uptake. This was determined as follows: Potassium phosphate
buffer (0.1 M) at pH 7.0 was added to the medium (1.5 ml/0.5 ml). The mixture was
extracted with chloroform (6 ml) by shaking for 90 sec. The chloroform and aqueous
phases were separated by centrifugation, the upper phase was removed and the
organic phase washed with 1 ml of potassium phosphate buffer, pH 7.0. The
radioactivity in a sample of the chloroform phase was measured. This method relies on
the fact that esterified lipids are not released in appreciable amounts to the incubation
medium, as shown by Whitelaw & Williamson (1976).
The radioactivity in ketone bodies was estimated by measuring the
radioactivity in acid-soluble compounds in the supernatant.
Calculation of rates: The time-courses of glucose synthesis from the
gluconeogenic precursors and of ketone body formation from long- and short-chain
fatty acids, in isolated liver cells, were linear between 20 and 60 minutes, and were
calculated as follows:
The animals (starved 48h post-operation) were anaesthetized with an
intraperitonial injection of pentobarbital (50mg/kg body wt). Each liver was removed,
minced and put into a pre-weighed ice-cooled 50 ml beaker containing 10ml of NaF
sucrose (0.25 M sucrose; 50 mM NaF) buffer. More NaF sucrose buffer was added to
the beaker to a final volume/liver wt. of (10:1). Livers were then homogenized using a
glass homogenizer kept in ice. After homogenization
samples were
centrifuged a
800 g (15 min), 16,000 g (20 min) and again at 16,000 g (20 min) to separate the
microsomes from the cell wall, red cells and cytosol during the first spin; from the
mitochondria and
the lysosomes during the second spin; and from the remaining
mitochondria and lysosomes during the third spin. The supernatant of the third spin
was carefully transferred to 12.5ml tubes and ultracentrifuged for 1 h at 110,000 g to
precipitate the microsomes. The supernatant was then discarded and the microsomes
were re-suspended in 20 mM imidazole/chloride - 5 mM dithiothreitol
assayed for HMGCoA reductase activity.
HMGCoA Reductase
Buffer solutions required for the assay:
Buffer A
20mM Imidazole /Chloride
(pH 7.4)
5mM Dithiothreitol
Buffer B
350mM Potassium phosphate buffer
40mM EDTA
20mM Dithiothreitol
buffer and
Cofactors
NADP+
10.5mg in 0.5ml H2O
Glucose-6-phosphate
33.5mg in 0.5ml H2O
Glucose-6-phosphate dehydrogenase
19U (50µl)+0.45ml H2O
These were mixed together. Buffer B (1.38ml) and H2O (0.38ml) were
added - The cofactor mixture
Microsomes, prepared as described previously, were suspended in 20mM
imidazole/chloride - 5 mM dithiothreitol (Buffer A) and incubated in duplicate in the
presence or absence of alkaline phosphatase at 37°C for 30 min , so that the total and
the expressed amount of enzyme activity could be measured. Before incubation
portions (10µl) of the homogenate were taken in duplicate to measure the protein
content. Three control incubations each containing buffer A instead of microsomal
homogenate. To the pre-incubated microsomes the cofactor mixture was added and a
solution of (R,S) [3-14C]HMG-CoA (80,000 dpm; 6.4 nmol; 8µl) in KH2PO4 (0.1 M)
was added to each tube giving a final (R,S)-HMG-CoA concentration of 145 µM. Each
tube was capped and placed in a water bath at 37°C. The rack in which the tubes were
placed was shaken at 70 oscillations per min. Afer 20 min each tube was removed
from the water bath and the reaction stopped by the immediate addition of HCl (12 M,
5µl).
An internal standard , consisting in a solution in water of (R,S)[2-3H
]mevalonolactone (5µl - 10,986 dpm), was added to each tube. The mevalonic acid
was lactonised by incubationg the tubes at 37°C for 30 min. At this stage , if necessary
, the samples could be stored for 1-3 days at -20°C.
Thin Layer Chromatography of Mevalonolactone
To precipitate protein , the tubes were centrifuged at 1000 g for 10 min.
Portions (40µl) of the supernatant of each sample were loaded into preadsorbent
strips of a multiple channel, glass-backed, Whatman LK5D silica gel TLC plate (20 X
20 cm; 0.8 cm wide channels). The plates were thoroughly dried for 4 h in a TLC tank
containing activated silica gel (coarse mesh). They were then developed with
ethylacetate/acetone (4:1,v/v) until the solvent, had reached 1 cm from the top of the
plate. The TLC tank contained 75 ml of the solvent, was lined with chromatography
paper and had been pre-equilibrated for at least 30 min. The plates were dried and the
mevalonolactone-containing band was
located by spraying with a solution of Rhodamine 6G
in acetone (0.1%,w/v) and
visualisation under ultra-violet light. This area of the plate was scraped from the plates
with a razor blade and transferred into scintillation glass vials. Ethanol (1 ml) was
added,
followed
by
10
ml
of
a
toluene-based scintillation cocktail
(PPO/POPOP/Toluene ; w/w/v ; 0.3 : 0.01 : 100). The 3H and 14C contents of the
sterol fraction were determined by scintillation counting for 30 min per sample.
Microsomal Protein Determination
The method of Lowry et al (1951) was used, as modified to assay protein in
the presence of sulphydryl reagents, by Geyger and Bessman (1972). The standard
curve was constructed using 0-150 µg of bovine serum albumin. After the microsomes
were re-suspended in 20mM imidazole/chloride - 5 mM thiothreitol (Buffer A) and were
ready for the incubation, a portion of the suspension from each sample (10µl) was
taken in duplicate into 10 ml tubes to measure protein. The dithiothreitol was oxidised
by heating in the presence of H2O2.
Reagents:
A. 1 % CuSO4 5 H2O
B.
5.4% NaK Tartratel
C.
10% Na2CO3 ; 0.5 M NaOH
Copper Reagent:
1 ml of reagent A was mixed with 1 ml of reagent B
1 ml of this new mix (A+B) was added to 10 ml of reagent C
Phenol Reagent:
The phenol reagent (F&C) was diluted with distilled water (v/v; 1:10)
Distilled water was added to the standards and the protein-containing tubes
up to a final volume per sample of 0.75 ml. The tubes were mixed and 0.75 ml of the
copper reagent added; 15 µl of 3% H2O2 were also added to each tube and they were
mixed again. The tubes were transferred to a water bath at 60°C for 10 min. Phenol
reagent (1.5 ml) was added to each sample and standard , mixed and each tube
transferred again to the water bath at 60°C for another 10 min. Tubes were cooled and
each standard and each sample was transferred to the same glass cuvette to be read
in the spectrophotometer at 750 nM. The standard curve was then constructed and the
concentration of protein in the samples calculated based on this standard curve.
Calculation of enzyme activity
The 14C dpm content of each sample was corrected for the recovery of [23H]mevalonolactone and by subtracting the average 14C dpm value obtained from the
control incubations (usually about 25 dpm). The corrected amount of 14C dpm was
then converted to pmol of mevalonolactone (the specific radioactivity of the substrate
used was 12.5 dpm/pmol).
Therefore, the following equation was used to calculate enzyme activity:
HMG-CoA reductase activity was expressed as pmol of mevalonolactone
produced per min per mg of microsomal protein and was the average of duplicate
assays.
Measurements of 3H and 14C radioactivity by liquid scintillation counting
were performed using an LS 9800 Liquid Scintillation System (Beckman Instruments
Inc., Irvine, CA, USA).
The sample to be measured was dissolved in a toluene-based scintillation
fluid (10 ml). Depending on the specific scintillation cocktail or radionuclide use,
distilled water and or ethanol was added to the vials. The vials were fitted with a screw
cap and when all the contents (sample, scintillation fluid and additions ; e.g. water,
ethanol) and were counted as described by Williamson et at (1975).
The counting procedure is based on the fact that ionising ß-particles
produced by the decay of 3H or 14C atoms lose some of their kinetic energy to the
scintillant. This energy is converted into photons. Initially the kinetic energy is absorbed
by the toluene and is subsequently transferred to the scintillant present in the
scintillation fluid. The photon emissions are detected as voltage pulses, proportional in
magnitude to the energy of the ß-particles, by two photomultiplier tubes. Only
coincident pulses are counted by the machine to eliminate random events.
The overall yield for a particular radionuclide will be reduced by several
factors, which are termed quenching. The level of quenching was monitored and the
counting energy channels optimised for each sample by an automatic quench
compensation method. When in the dual-label counting mode (3H-14C), the machine
was calibrated with a series of differently quenched samples containing either 3H or
14C of known dpm. The energy channels were set such that
there was no spillover from 3H into 14C. The quenching data and shannel setting
during either single or dual-lable counting, were stored in the instrument as a 'user
counting program'. A microprocessor built into the machine automatically calculated the
3H and/or 14C dpm using this program.
Experimental results were expressed as the mean ± the standard error of the
mean (S.E.M.) accompanied by the number of observations (n). The S.E.M. is
mathematically defined as:
Results were analysed using the Student t-test.
CHAPTER 2
MATERIALS AND METHODS
[1-14C]Oleate, [14C]HMGCoA, [3H]mevalonolactone and 3H2O were
purchased from the Amersham International, Amersham, Bucks, U.K. Others
biochemicals and enzymes were obtained from Boehringer Corp. (London)Ltd.,
Lewes, Sussex, U.K.
Animals
Male rats of the Wistar strain weighing about 250g were fed ad libitum
on a breeding diet for rats and mice (Special Diet Services, Witham, Essex,
U.K.) containing 4% w/w lipid, 21% crude protein and 52% carbohydrate; the
residue was non-digestible material. The animals were maintained at an ambient
temperature of 22±2°C under a 12h light/12h dark cycle (lights on 07.30h).
Operations were carried out under light ether anaesthesia. A midline
laparotomy was performed. The caecum was mobilized by incising the
mesocaecum and the faeces were milked into the caecum, which was then
ligated with a single 3-0 silk ligature in
such a manner that bowel continuity was maintained (Fig. 2.1. & 2.2.).
Fig. 2.1.
The anti-mesenteric surface of the caecum was punctured once with a 21-gauge
needle, and the bowel returned to the abdominal cavity (Fig. 2.2.).
Fig. 2.2.
The abdominal wall was closed in two layers and all rats received 0.9% (v/v)
NaCl (2.5ml/100g body wt.) subcutaneously. With this procedure an
experimental peritonitis is created (Wichterman et al , 1980) with a microbial
flora that closely approximates that of human peritonitis (McGowan &
Gorbach, 1983) and therefore has proven to be a suitable model of the human
disease. Water was offered ad libitum .
The rectal temperatures of the septic rats were not different to those of
the sham-operated group. It has been reported that blood pressure of rats made
septic by this procedure is unchanged (Wichterman et al , 1980). The single
puncture with a small needle gauge (21 G) was chosen so that the peritonitis
could develop at a slower rate allowing animals to starve and survive up to 48 h.
The mortality rate was 3.7% (one out of 27) at 12 h, 5.5% (two out of 36) at 24
h and 18.9% (15 out of 79) at 48 h , which was lower than that observed by
Wichterman et al (1980) using a single puncture operation.
Sham-operated rats
In the sham-operated animals the caecum was
also mobilized by
incising the mesocaecum and then immediately returned to the abdominal
cavity (Fig. 2.3.). All the other steps of the operation and post-operative care
were similar to those described for the caecum-ligated animals. Sham-operated
rats recovered more promptly from the anaesthesia and did not show any of the
signs of sepsis, as described by Wichterman et al (1980), at the various times
they were studied.
Fig. 2.3.
A group of animals was subjected to total interruption of their bile acid
hepato-enteric circulation. In these animals, after the midline laparotomy was
performed, all the small bowel was mobilized up to the duodenal-jejunal
junction so that the bile duct entry into the duodenum could be visualized. The
bile duct was isolated at approximately one centimeter from the entry point and
ligated with a single 3-0 silk ligature . The caecum was then mobilized and all
the other steps of the operation and the post-operative care were the same as to
those in either experimental (caecum-ligated and punctured) or control (shamoperated) animals.
Rats (starved 12, 24 or 48h post-operation) were anaesthetized with
pentobarbital (50mg/kg body wt.), injected intraperitoneally, and a laparotomy
performed. Arterial blood from the abdominal aorta was collected using
heparinized syringes (2 ml) and the liver removed. Part of the latter was
immediately freeze-clamped (Wollenberger et al , 1960). This was achieved by
freezing the tissue between aluminium tongs cooled in liquid nitrogen (i.e. at a
temperature of about -190°C) which lowered the temperature of the tissue to
about -80°C in less than 0.1 sec. If not extracted for metabolite determination on
the same day, freeze-clamped livers were kept stored in aluminium foil in a
liquid nitrogen-refrigerator tank. To measure hepatic metabolites freezeclamped livers were ground to a fine powder with liquid nitrogen-cooled
porcelain mortar and pestle (Williamson et al , 1967) and extracted in icecooled 0.75M HClO4. This rapidly inactivated and precipitated protein which
was removed by centrifugation at 15000 rpm for 20 min. This HClO4 extract
was then neutralised with a 20% solution of potassium hydroxide (KOH)
(Analar,
BDH)
and
the
metabolic
intermediates
measured
by
spectrophotometric methods. The remaining part of the liver was used for
measurements of lipid and cholesterol synthesis or determination of enzyme
activity. Metabolites were extracted from total blood with 0.75M HCl04 as
previously described by Williamson et al (1967). The tubes containing blood
and HClO4 were centrifuged at 3000 rpm for 10 min. The supernatant was
decanted into pre-weighed labelled 10ml plastic tubes which were then
reweighed. One drop of universal indicator (BDH) was added to the supernatant
followed by a 20% solution of potassium hydroxide (KOH) (Analar, BDH),
which was added drop-wise until the pH was between 7 and 8. After allowing to
stand in ice for 10 min, the tubes were reweighed and centrifuged at 3000 rpm
for 10 min, and the supernatant, now a neutral extract, was used for metabolite
analysis. Pyruvate and acetoacetate were measured immediately after
neutralisation and the other metabolites were measured on the same day of
sampling or after storage at -20°C within two weeks. Part of the total blood
sample was centrifuged at 3000 rpm for 10 min and the plasma was separated
and used for determination of free fatty acids, triacylglycerol and insulin
concentrations and measurement of the specific radioactivity of plasma water.
Before giving details about the various enzymatic methods used to
determine metabolite concentrations, the principles behind enzymatic analysis
are introduced.
The enzymatic assay of metabolic substrates is based on the principle
that a specific enzymatic reaction in which the substrate participates is coupled
with the reduction of NAD/NADP or oxidation of NADH/NADPH. The
pyridine nucleotides (NAD, NADP) absorb light at 260 nm, and in the reduced
state (NADH, NADPH) they have an additional absorption band with a
maximun at 340 nm. By measuring the optical density at 340 nm, the enzymatic
conversion of the substrate can be followed directly in a spectrophotometer
cuvette. Regardless of whether NAD accepts H+ or whether NADH donates
H+, at this wavelength the optical density increases or decreases by 6.22 units
(light path 1 cm) with the production or consumption of 1 µmole of
NADH/NADPH. Since in a specific enzymatic reaction, 1 µmole of substrate
usually co-reacts with 1 µmole of NAD/NADP (or NADH/NADPH), the
change in optical density will reflect accurately the amount of substrate
consumed by the reaction. Provided the assay conditions are optimum,
conversion of the substrate is practically complete and the optical density
difference (ODD) can be used to calculate the concentration of the substrate in
the blood or tissue sample by multiplying with an appropriate dilution factor.
Since many enzymatic reactions are equilibrium reactions, in order to
make an end-point measurement the equilibrium of the reaction has to be
displaced such that it favours the complete consumption of the substrate. The
reaction equilibrium can be influenced by several factors such as increase in
substrate or cofactor concentration, variation of pH, presence of trapping agents,
or the use of regenerating reactions in which one of the co-substrates may be
regenerated by a secondary reaction. If none of the reactants or products of an
enzymic reactions is measurable spectrophotometrically, it is often possible to
transform one of the products by another enzymatic reaction which in turn can
be easily measured (e.g., measurement of glucose, non-esterified fatty acids and
triacylglycerols). The former reaction, in which the substrate to be determined is
transformed, is known as the auxiliary reaction, whereas the reaction used for
the actual measurement is known as the indicator reaction. Both reactions can
usually be carried out in the same assay mixture (Bergmeyer,1963).
Specificity of an enzymatic assay depends on the purity of the enzyme
preparation whereas precision depends on the provision of optimum assay
conditions. The sensitivity of enzymatic assays is limited by the fact that
sufficient conversion of NAD/NADP (or NADH/NADPH) must occur to
produce a measurable change in the optical density.
The following metabolites were measured by standard enzymatic methods as
described below:
Determination of D-Glucose
Blood and liver glucose were measured according to the method
described by
Slein (1963).
Reaction sequence:
a. Auxiliary reaction
GLUCOSE
+
Mg++
ATP
——————————————>
GLUCOSE-6-
PHOSPHATE + ADP
Mg++
glucose-6-phosphate
GLUCOSE-6-PHOSPHATE
dehydrogenase
6-
——————————————————————>
PHOSPHOGLUCONATE
+ NADP+
+ NADPH + H+
At pH 7.5, the equilibrium for the indicator reaction is far to the
right
which
ensures the completion of both reactions (since
glucose-6-phosphate
formed in the former is rapidly used up in the
latter
Although hexokinase catalyses the phosphorylation
of
monosaccharides, specificity is provided by
glucose-6-phosphate
dehydrogenase (G6PD) with which hexose or
pentose esters other
than glucose-6-phosphate do not react.
reaction).
several
other
20ml 0.1M tris buffer pH 8.0
2ml 0.1M magnesium chloride
2ml 0.01 M ATP
2ml 1% NADP
0.13ml of G6PD (1mg/ml)
This was prepared freshly for each assay. The total volume in each
cuvette was
2.0ml. In the sample cuvettes, this consisted of 0.1ml of
neutralised
HClO4
extract, 0.9ml of distilled water and 1ml of assay
buffer; in the control
cuvette 1ml of water and 1ml of assay buffer
was
cuvettes were read at 340nm before , at 10min and
15min after addition
added.
The
of 0.010ml of hexokinase.
Measurement of L-(+)-Lactate
Concentrations of blood and liver lactate were measured according to
the
method
described by Hohorst (1963).
Reaction sequence:
Lactate dehydrogenase
LACTATE + NAD+
——————————————> PYRUVATE +
NADH + H+
The equilibrium of this reaction lies well on the side of lactate and
NAD.
Therefore, in order to ensure the complete conversion of
lactate, the reaction
products have to be removed from the
equilibrium.
are trapped by an alkaline reaction medium;
the pyruvate reacts
with hydrazine hydrate in the
buffer
form pyruvate hydrazone and, in addition, a large excess of
solution
to
NAD and enzyme is
used to obtain a sufficiently rapid end point.
Lactate
dehydrogenase reacts only with L-(+)-lactate and thus
provides
for the assay.
Protons
specificity
40ml 0.2 M Tris
25mg EDTA
Made up to 100ml of distilled water.
The pH of the buffer solution was adjusted to pH 9.5 with 5M hydrochloric acid and
it was stored for up to two weeks at 4°C. Before
use, 1 ml of 1% (w/v)
NAD was added to every 10 ml of assay buffer.
The total volume in
each cuvette was 2.0ml. In the sample cuvettes
0.2ml of neutralised HClO4 extract, 0.8ml of water and
this
consisted
of
1ml of assay buffer;
the control cuvette contained 1ml of water and
1ml of assay buffer.
All cuvettes were read at 340 nm before and at 35
and 45 min after the
addition of 0.02ml of lactate dehydrogenase.
Measurement of Pyruvate and Acetoacetate
Pyruvate and acetoacetate share very similar assay conditions and
therefore
were measured sequentially in the same sample (same
cuvette) according to
a combination of methods respectively
described by Hohorst
et al (1959) and Williamson et al (1962).
Reaction sequence:
PYRUVATE + NADH + H+
————————————————————>
LACTATE + NAD
3-OH-butyrate dehydrogenase
ACETOACETATE
+
NADH
HYDROXYBUTYRATE + NAD+
+
H+
—————————————>
The equilibrium of the first reaction at pH 7.0 is sufficiently far to
3-
the right to
ensure a quantitative measurement of pyruvate levels
provided
that
the
NADH concentration is not less than 0.01 mM. At the
same pH and with a
suitable excess of NADH, at least 98% of the
acetoacetate
reduced to D-(-)-3-hydroxybutyrate. However, due to
the low activity of 3-
is
hydroxybutyrate dehydrogenase preparations
the second reaction
proceeds at a much slower rate than the former.
10ml of 0.1 Potassium phosphate buffer pH 6.9
1 ml of 0.5% (w/v) NADH
A fresh solution was prepared for each assay. The total volume in
each cuvette
was 2 ml. In the sample cuvettes, this consisted of 1ml of
neutralised
HClO4
extract and 1ml of assay buffer. The control
cuvette contained 1ml
of water and 1ml of assay buffer solution. The
cuvettes were read at
340 nm before and 10min after the addition of
of 0.01ml of lactate
dehydrogenase. 3-Hydroxybutyrate
dehydrogenase
(0.01ml) was then added to each cuvette and they
were read again at 35
and 45 min thereafter.
Measurement of D-(-)-3-hydroxybutyrate
Blood and hepatic concentrations of 3-hydroxybutyrate were measured
to the method described by Williamson et
according
al (1962).
Reaction sequence:
3-hydroxybutyrate dehydrogenase
3-OH-BUTYRATE + NAD+ ————————————>
NADH + H+
The equilibrium of the reaction at pH 8.0 is reached when
ACETOACETATE +
approximately 40% of
the 3-hydroxybutyrate is oxidised to
acetoacetate.
However, the presence of hydrazine in the buffer
solution
acetoacetate formed as a hydrazone and the
reaction
quantitatively from the left to the right.
traps
the
proceeds
70 ml 0.1M tris buffer pH 8.5
0.25 ml of hydrazine hydrate 100%
25mg of EDTA
The pH of the assay buffer solution was adjusted to pH 8.5 with 5M
hydrochloric
acid and it was stored for up to two weeks at 4°C. Before
use, 1 ml of 1% (w/v)
NAD was added to 10ml of assay buffer for each
assay.
volume in each cuvette was 2 ml. In the sample
consisted of 0.5 ml of neutralised HClO4 extract, 0.5ml of
cuvettes,
this
water and 1 ml of
assay buffer; the control cuvette contained 1 ml of
water and 1 ml of
assay buffer. The cuvettes were read at 340 nm
before and 50 and 60
min after the addition of 0.01 ml of
3-hydroxybutyrate
The
total
dehydrogenase.
Measurement of L-Alanine
Blood and hepatic concentrations were measured according to the
method
described by Williamson et al (1967).
Reaction sequence:
alanine dehydrogenase
ALANINE + H2O + NAD+ ——————————————> PYRUVATE + NH4+
+ NADH
In the presence of low H+ ion concentration, at pH 9.0, this reaction
proceeds
quantitatively from the left to the right. Hydrazine hydrate
is included in the
buffer solution in order to trap the pyruvate
formed by conversion
to pyruvate hydrazone.
40ml 0.2M tris buffer
25mg EDTA
The pH of the buffer solution was adjusted to pH 9.0 with 5 M
hydrochloric
acid and it was stored for up to 14 days at 4°C. Before
use, 1ml of 1% (w/v)
NAD was added to 10ml of assay buffer for each
assay.
The
total
volume per cuvette was 2 ml. In the sample cuvettes,
ml of neutralised HClO4 extract,0.5 ml of water
this consisted of 0.5
and 1 ml of assay
buffer; the control cuvette contained 1 ml of water
and 1 ml of asssay
buffer.All cuvettes were read at 340 nm before and
at 50 and 60 min after
addition of 0.01ml of alanine dehydrogenase.
Determination of D-3-Phosphoglycerate,
D-2-Phosphoglycerate and Phosphoenolpyruvate and
Pyruvate
Hepatic concentrations of pyruvate, PEP, 2-phosphglycerate and
phosphoglycerate were measured according to the method
3-
described by Czok &
Eckert (1963).
Reaction sequence:
a.
PYRUVATE
LACTATE + NAD+
+ NADH + H+
-——————————————>
pyruvate kinase
b.
PHOSPHOENOLPYRUVATE
+
ADP
———————————>
PYRUVATE + ATP
Mg2+, K+
enolase
c.
2-PHOSPHOGLYCERATE
———————————>
PHOSPHOENOLPYRUVATE
Mg2+
phosphoglycerate mutase
d.
3-PHOSPHOGLYCERATE
PHOSPHOGLYCERATE
—————————————>
2-
At pH 7.4 quantitative conversion of the substitute is assured
because
of
positions of the equilibrium state of the reactions
catalysed by pyruvate
kinase and lactate dehydrogenase being far to
the
Phosphglycerate can only be determined under the
here if the assay mixture contains less than 10-3
conditions
described
M
inorganic
phosphate. The determination of 2-phosphoglycerate,
phosphoenolpyruvate
and pyruvate is not affected by phosphate.
Specificity is secured
by the high specificity of the reactions
catalysed by enolase
right.
the
3-
and pyruvate kinase.
10 ml
Tris HCL buffer ph 7.4 0.1M
1 ml
Mg Cl2
3 ml
KCl 1.0 M
1 ml
ADP 0.02 M
0.5 ml
0.1 M
NADH 0.5 %
This was prepared freshly for each assay. The total volume in each
cuvette was
2.0 ml. In the sample cuvettes, this consisted of 0.5 ml of
HClO4 extract, 0.5 ml of distilled water and 1 ml of
neutralised
liver
assay buffer; in the
control cuvette 1 ml of water and 1 ml of assay
buffer
The cuvettes were read at 340 nm before and for
5-10 min
addition of lactate dehydrogenase (0.01ml);
pyruvate kinase (0.01
ml) was added and the changes in the optical
density followed for
5-10min; enolase (0.01ml) was added and the
cuvettes
continuously for 5-10 min; finally
phosphoglycerate
mutase (0.01 ml) was added to the cuvettes and the
change in the optical
density followed for 15-20 min. All cuvettes
were read using a
Gilford recording spectrophotometer.
were added.
after the
were
read
Measurement of Dihydroxyacetone Phosphate,
D-Glyceraldehyde-3-
phosphate and
Fructose-
1,6-bisphosphate
Hepatic
concentrations
of
dihydroxyacetone
phosphate
glyceraldehyde 3-phosphate (GAP) and fructose 1,6-bisphosphate
determined acording to the method described by Bücher &
(DHAP)
,
were
Hohorst (1963).
Reaction sequence:
GDH
DHAP +NADH + H+ —————> L-(-)GLYCEROL-1-PHOSPHATE +
a.
NAD+
TIM
b.
GLYCERALDEHYDE -3-PHOSPHATE (GAP) ——> DIHYDROXYACETONE
PHOSPHATE
aldolase
c.
FRUCTOSE-1,6-BISPHOSPHATE (FDP) ———————> GAP + DHAP
The equilibrium state of the reactions ensures that the overall reaction
proceeds
quantitatively. For each mole of FDP formed 2
moles of NADH are
oxidised, therefore, the inclusion of the
triosephosphate
isomerase (TIM) reaction increases the sensitivity of
the assay.
10 ml
Tris HCl ph 7.4
1 ml
NADH 0.5%
The total volume in each cuvette was 2 ml. In the sample cuvettes,
this
consisted of 1 ml of neutralised liver HClO4 extract and 1 ml of
assay buffer solution;
the control cuvette contained 1 ml of distilled
water and 1 ml of
assay buffer. All cuvettes were read continuously
using
spectrophotometer as follows: before and for 5-10 min
after
glycerol-3-phosphate dehydrogenase (GDH); before
and for 5-10 min after
addition of triophosphate isomerase (TIM) and
before and for 10-15
min after addition of aldolase.
a
Gilford
addition
of
Measurement of Adenosine-5'-Triphosphate and
D-glucose-6-
phosphate
Adenosine-5'-triphosphate (ATP) and glucose -6-phosphate were
measured
with a combination of the methods described by
Lamprecht
Trautschold (1963) and Hohorst (1963); using the same
cuvette.
&
phosphorylates glucose with ATP in the
Hexokinase
presence of Mg++ to
give glucose-6-phosphate. Glucose-6-phosphate
dehydrogenase
catalyses the oxidation of glucose-6-phosphate with
NADP. Each mole of
ATP forms 1 mole of NADPH.
Reaction sequence:
hexokinase
GLUCOSE + ATP ——————————> GLUCOSE-6-PHOSPHATE (G-6-P) +
ADP
G6P-DH
G-6-P + NADP+ ———————> 6-PHOSPHOGLUCONO-LACTONE + NADPH +
H+
In the presence of equivalent concentrations of glucose and Mg2+,
virtually quantitatively converted to ADP by hexokinase. The
ATP
equilibrium
is
of
the
glucose-6-phosphate reaction lies to the right.
10 ml
Tris
0.1M
pH 7.4
1 mg/ml NADP
2 ml
Mg Cl2 0.1 M
0.5 ml
Glucose
0.1 M
This was prepared freshly for each assay. The final volume per
cuvette was
2 ml. In the sample cuvettes this consisted of 0.5 ml of
liver HClO4 extract,
0.5 ml of distilled water and 1 ml of buffer
solution. The control
cuvette contained 1 ml of distilled water and 1
ml
solution.Cuvettes were read at 340 nm before and 5-10
min after 0.005 ml of
glucose-6-phosphate dehydrogenase was added
and subsequently 10-
15 min after hexokinase (0.005 ml) was added.
of
buffer
Determination of Acetyl-Coenzyme A
Hepatic acetyl-CoA was measured by the method described by Decker (1963).
Reaction sequence:
citrate synthase
ACETYL-SCoA + OXALOACETATE + H2O ———————> CITRATE + CoASH + H+
L-MALATE + NAD+ ————————————————> OXALOACETATE +
NADH + H+
At pH 8.0 the equilibrium of the over-all reaction lies to the right and
acetyl-CoA
is used quantitatively.
Buffer solution used for assay:
15 ml Tris 0.1 M
1 ml
L-malate
0.05 ml NADH
1 ml NAD
1 ml
pH 8.0
0.1 M
0.5 %
1%
This was made freshly for each assay. The total volume per cuvette
was 2 ml. In
the sample cuvettes this consisted of 1 ml of liver HClO4
extract and 1 ml of
buffer solution. The control cuvette contained 1
ml of distilled water
and 1 ml of buffer solution. Cuvettes were read
before and at 15 min
after addition of citrate synthase (0.01 ml).
Determination of L-Glutamate and Glutamine
Hepatic glutamate was measured according to the method described
Bergmeyer (1963). The determination of glutamine was
enzymatic technique.
Reaction sequence:
glutamate dehydrogenase
by Bernt &
also based on this
L-GLUTAMATE + NAD+————————————————> OXOGLUTARATE +
NADH + NH4+
glutaminase
L-GLUTAMINE –––––––––––––––––––––––––> L-GLUTAMINE + NH4+
The equilibrium lies far to the left. However, under alkaline
conditions
and the presence of excess NAD, oxoglutarate can
be
hydrazine and glutamate quantitatively oxidized to
oxoglutarate.
(pH 9.0)
trapped
with
2 ml Hydrazine hydrate 100 %
20 ml Tris 0.2 M
25 mg EDTA
Made up to 40 ml with distilled water and the pH (9.0) adjusted using 5
N
HCl. This could be kept at 4°C for up to 14 days. To be used for assay
(cocktail), NAD (1mg/ml) and ADP (0.5mg/ml) were added to the
buffer
solution. The total volume per cuvette was 2 ml. In the sample
of 1 ml of liver HClO4 extract and 1 ml of
cuvettes this consisted
cocktail. The control
cuvette contained 1 ml of distilled water and 1
ml
Cuvettes were read before and 30-40 min after the
addition of glutamate
of
cocktail.
dehydrogenase (in glycerol).
Glutamine
In order to measure glutamine liver HClO4 extracts were
presence of glutaminase under the contitions
0.5 ml
pre-incubated in the
described below:
Liver extract
0.5 M Acetate buffer 0.5 M
pH 4.8
0.01 ml NH2OH
1M
0.02 ml Glutaminase [5 mg/ml]
The total volume per tube was made up to 1 ml with distilled water. An internal
standard was added, consisting of 1 mM glutamine (0.2ml)
and
distilled
water
(0.8 ml). Tubes were mixed and incubated at 37°C
for 45 min. These
were then cooled, 0.5 ml of the mixture taken and
the concentration of
glutamate measured by the enzymatic method
described
above
(Bernt & Bergmeyer, 1963). As glutamine is
converted
to
glutamate by glutaminase, the total concentration of
glutamine+glutamate
is measured spectrophotometrically. Therefore,
the
actual
concentration of glutamine is calculated by subtracting
the concentration of
glutamate measured in the liver extract from
the total concentration
(glutamine + glutamate) found in
the glutaminase pre-
incubated extract.
Measurement of Non-esterified fatty acids
The method of Shimizu et al (1979) was used for the measurement of nonesterified fatty acids (NEFA), which is based on the activation of
NEFA by acyl-CoA
synthetase.
Reaction sequence:
acyl-Co A synthetase
NEFA + COENZYME A + ATP —————————> ACYL COENZYME A +
AMP+ PPi
myokinase
AMP + ATP ———————> 2 ADP
pyruvate kinase
2 ADP + PHOSPHOENOLPYRUVATE
————————->
2 ATP + 2
PYRUVATE
2 PYRUVATE + 2 NADH ————————————————> 2 LACTATE
+ 2 NAD+
The reaction calalysed by acyl-CoA synthetase favours the
production
of
acyl
coenzyme A provided the pH is 8.0 and there are
sufficient amounts of
ATP and coenzyme A. Therefore, the production
of AMP is used as a
measure of NEFA activation through an indicator
system provided by
successive reactions catalysed by myokinase,
pyruvate kinase and
lactate dehydrogenase. The equilibrium state
of all three reactions
in this indicator system is sufficiently far to
the right to ensure that
the overall rate of the reaction is limited
only
concentration of NEFA. Specificity for the method is
enhanced by the fact
that Acyl CoA synthetase only activates
monocarboxylic acids
by
the
with 6 to 18 carbon atoms and does not affect
other carboxylic acids
and lipids.
200 ml 0.1 M tris pH 8.0
35.1 mg EDTA (0.6 mM)
406.6 mg magnesium chloride (10mM)
2 ml Triton X-100
This was stored at 4°C. Immediately before each assay, the following
reagents
were added to 36 ml of buffer solution:
1.6 ml NADH 0.5% (w/v)
1 ml
ATP 0.1 M
1 ml phosphoenolpyruvate 0.2 M
0.2 ml myokinase 2mg/ml
0.4 ml pyruvate kinase 1mg/ml
0.2 ml lactate dehydrogenase 10mg/ml
The total volume per cuvette was 2.005 ml. The sample cuvettes
contained
0.04ml of plasma, 1.95ml of buffer mixture and 0.015ml of
acyl-CoA synthetase.
The plasma volume was replaced by 0.04ml of
water in the control
cuvette. All cuvettes were read at 340 nm before
and 20 min after the
addition of coenzyme A. Further readings were
taken
intervals until the readings of the optical density
were constant.
at
5
min
Measurement of Triacylglycerol
Plasma triacylglycerol concentration was measured according to the
method
described by Eggstein & Kreutz (1966). Plasma triacylglycerol
were
hydrolysed to glycerol and non-esterified fatty acids.
The glycerol
measured by the enzymatic techique described
concentration of free glycerol, measured in the
below.
neutralised
extract was subtracted from the total glycerol
measured
alkaline hydrolysis of the plasma sample to obtain the
glyceride-glycerol
concentration which, in turn, was taken to be an
estimate
triacylglycerol content of the plasma.
Reaction sequence:
a. Hydrolysis:
at 70°C
initially
was
The
HClO4
after
of
the
TRIACYLGLYCEROL + 3 H2O ————————> GLYCEROL + 3
FATTY ACIDS
alchoholic KOH
glycerokinase
pyruvate kinase
GLYCEROL + PHOSPHOENOLPYRUVATE
——————> GLYCEROL-3-PO4 +
LACTATE
ATP
ADP
NADH
ATP
NAD+
Hydrolysis:
To 0.2 ml of plasma 0.5ml of alcoholic KOH (0.5N KOH in 98% ethanol)
was
added. The mixture was incubated at 70°C for 30 min and then
1.5ml of 0.1 M MgSO4 was added to precipitate
cooled.
protein,
samples were centrifuged at 3000 rpm for 10 min.
The
present in plasma produced equimolar quantities
of
glycerol produced by hydrolysis was contained in
the
solution, 0.5ml of which was used for the enzymatic
measurement of total
glycerol.
Thereafter,
and
the
triacylglycerol
glycerol.
This
supernatant
Measurement of glycerol
Reaction sequence:
glycerokinase
GLYCEROL + ATP ———————————> GLYCEROL-3-PO4 +
ADP
pyruvate kinase
ADP
+
PHOSPHOENOLPYRUVATE
—————————>
ATP +
PYRUVATE
PYRUVATE + NADH + H+ ————————————> LACTATE +
NAD+
The equilibrium state for all three reactions can be acheived at pH
the presence of Mg++ ions. It is far to the right to ensure
that
7.4 and in
there
is
a
quantitative consumption of glycerol in the first
reaction and that the
indicator reactions can rapidly proceed to
completion.
30 ml 0.1 M tris buffer pH 7.4
3 ml 0.1 M magnesium chloride
35 mg phosphoenolpyruvate
50 mg ATP
12.5 mg
NADH
0.2 ml lactate dehydrogenase
0.2 ml pyruvate kinase
This was made up immediately before use and the pH adjusted to pH
7.4 with 0.2
M tris buffer.
Triacylglycerol
The total final volume per cuvette was 2 ml. In the sample cuvettes,
hydrolysed plasma sample was added to 1 ml of assay
0.5ml of the
buffer and 0.5 ml of
distilled water.
Glycerol
The sample cuvettes contained 1 ml of assay buffer and 1 ml of
neutralised
HClO4 extract. Blank cuvettes contained 0.5 ml of buffer
mixture and 1.5 ml of
distilled water. The control cuvette contained 1
ml of distilled water
and 1 ml of assay buffer. All cuvettes were read
at 340 nm before and
40 min after addition of 0.01 ml of
glycerokinase.
were then re-read every 10 min until there was
no further change in
the optical density.
They
Calculations for blood and liver metabolites
All metabolite calculations were based on the change in optical density
measured at 340 nm in the sample cuvettes following addition of enzyme, and
after subtraction of the non-specific change which
occurred in the control
cuvettes.
Therefore:
Optical density difference (ODD) = Change in absorbance for sample cuvette - Change in
absorbance for control cuvettes
Since the molar extinction coefficient of NADH is 6.22 cm2/µmol, the amount of substrate in the
cuvette = ODD / 6.22 X total volume in cuvette.
This is multipied by the dilution factor for each sample to give the concentration of substrate;
In the blood:
In the liver: 1 g frozen liver = 0.8 ml ; 1 g liver + 4 ml PCA = 4.8
Insulin Determination
Insulin was measured by radioimmunoassay with a rat insulin standard
(Albano et al , 1972) using activated charcoal for separation of the free and bound
hormone. A disequilibrium assay was set up with a 3-day incubation period and
all plasma samples were assayed in duplicate with 20 µl of plasma in each tube.
The essential principle of the insulin asay, as for any other radioimmunoassay
technique, is the reaction of a fixed amount of specific insulin antibody with a
mixture of the plasma sample to be assayed and a constant amount of
radioactively labbelled pure insulin. The reaction is initiated by incubating the
plasma sample with insulin antibody for approximately 48 hours. The
radioactively labelled insulin is then added to compete for the remaining binding
sites on the antibody complex. The reaction is allowed to approach completion
over the subsequent 24 hours, and the antibody-bound insulin is separated from
the free hormone by differential adsorption on to albumin-coated charcoal. The
distribution of the radioactivity between the free and bound forms of insulin is
then determined. It is expected that the amount of radioactively labelled insulin
bound to antibody to decrease as the concentration of the unlabelled insulin in the
plasma sample increases due to competition for the limited number of binding
sites available. Therefore, results obtained with samples of unknown insulin
concentration can be compared with standard curves obtained by the
measurement of samples to which known amounts of pure insulin standard have
been added.
Lipogenesis and cholesterolegenesis were measured in vivo with 3H2O as
previously described ( Jungas et al , 1968; Robinson et al , 1978). The rats were
injected intraperitoneally with 5 mCi (0.25ml) of 3H2O, and tissues removed one
hour later; 5 min before removal of tissue the rats were anaesthetized with
pentobarbital (50mg/kg body wt.). Heparinised blood was collected for
measurement of the specific activity of plasma water. The tissues (in duplicate
into 50 ml tubes) were hydrolysed under alkaline conditions by addition of 3 ml
of 30% KOH and heated in a water bath at 70°C for 15 min. Absolute alcohol
was then added and the samples returned to the 70°C water bath for a further 175
min (Stansbie et al , 1976). The non-saponifiable lipids were extracted three times
with light petroleum (b.p. 40-60°C; total vol. 20 ml) and the organic extract was
washed twice with distilled water and transfered to scintillation vials. After
evaporation of the solvent, the 3H radioactivity of the residue was measured by
scintillation counting in half of the samples to determine the total incorporation of
3H O into the non-saponifiable fraction. Cholesterol and squalene were isolated
2
from the non-saponifiable residue, from the other half of the samples, by
chromatography on thin-layer plates of silica gel H
developed with
chloroform/methanol (99:1, v/v). The plate was dried and the cholesterolcontaining band was located by spraying with a solution of Rhodamine 6G in
acetone (0.1%,w/v). This area of the plate was scraped into a scintillation vial,
and the 3H content of the sterol fractions were determined by scintillation
counting (Gibbons et al , 1983).
The aqueous phase after extraction of the non-saponifiable lipid was
acidified to pH 1.0 with 6N H2SO4, the labelled fatty acid fraction was extracted
three times with light petroleum and the organic extract washed twice with
distilled water. The extract was transferred into vials to dry (Stansbie et al ,
1976) . After evaporation of the solvent, the 3H radioactivity of the residue was
determined by scintillation counting.
Preparation of isolated hepatocytes
Hepatocytes were prepared essentially as described by Berry & Friend
(1969) with the modifications described by Krebs et al (1974). Routine tests
indicated that about 90% of the hepatocytes excluded Trypan Blue.
Operative technique, liver perfusion and isolation of hepatocytes
The rat was anaesthetized by an intraperitoneal injection of pentobarbital
(50 mg/kg body wt.). Heparin (150 units; 0.15 ml) was then administered via a
saphenous vein. A laparotomy through a vertical mid-line incision was
performed. Two mid-transverse incisions were made to the left and the right of
the mid-line to allow an extended exposure of the abdominal cavity. The portal
vein and the descending vena cava were exposed by moving the intestines to the
left side of the abdominal cavity. One loose silk ligature (3-0) was placed around
the descending vena cava immediately above the right renal vein, and two
ligatures, about 1 cm apart, were placed around the portal vein. The portal vein
was then cannulated using a 17 gauge metallic intravenous cannula. After
withdrawal of the inner needle, the cannula was secured by tying the ligature that
was closest to the liver. The other tie was tightened to prevent blood from flowing
out of the lower part of the portal vein and to tie off the common bile duct. The
cannula was therefore firmly in place and the appearance of blood back-flow at
the cannula end from the penetrated portal vein confirmed the success of the
cannulation.
The thorax was immediately opened, through a transverse incision. A
loose ligature was placed around the inferior vena cava just below the heart. A 16
gauge plastic cannula was used to cannulate this vessel through the right atrium
and secured with one 3-0 silk tie.
Liver perfusion and treatment with collagenase:
The method of liver perfusion was that described by Hems et al
(1966). A water-jacketed oxygenator (Fig. 2.4.) was used and the temperature
kept approximately at 38°C. The perfusion medium used was the saline of Krebs
& Henseleit (1932) from which calcium was omitted because it decreases the
activity of the enzyme. The total volume of the perfusion fluid was initially 150
ml. The first 40 ml of the medium passing through the liver were discarded in
order to remove the bulk of the blood cells. At this point 30 mg of the crude
collagenase (Boehringer Corporation, London), suspended in a small volume of
perfusion medium were added. Pure collagenase is not suitable because the
contaminating proteinases in the crude preparation play a very important role. The
flow rate was approximately 25 ml /min. The perfusion medium was kept in
equilibrium with O2/CO2 (95:5 ; v/v). The perfusion was continued for 20-30 min
and any leaked perfusion fluid appearing in the abdominal
cavity was collected and returned to the reservoir (Fig. 2.4.). The action of the
enzyme was reflected by the degree of leaking; when this had reached a rate of
about 10 ml/min, usually after 25 min, the perfusion was discontinued and the
liver was transferred to a beaker. The medium used in the perfusion was added to
the broken-up liver tissue as this was put into a nylon cloth (mesh size 0.44 X
0.44 mm) which was on the top of a 100 ml disposable plastic beaker.
Fig. 2.4.
The liver tissue was then gently broken apart with a pair of scissors and the cells
teased out with a plastic spatula, always keeping the perfusion medium (which
already contained cells washed out from the liver during the perfusion) flowing
through the nylon cloth. The filtrate was centrifuged at 50 g for 2 min and the
cells were re-suspended in about 30 ml of the Ca++-containing Krebs-Henseleit
medium. The suspension was centrifuged again at 50 g for 2 min and the pellet
weighed and resuspended in 16 volumes of the Krebs-Henseleit medium
containing Ca++, when it was ready to be incubated. Isolated hepatocytes were
used immediately after preparation.
The isolated hepatocytes were incubated with constant shaking, at 65
oscillations per min, for 20, 40 or 60 minutes in Krebs-Henseleit saline (Krebs &
Henseleit, 1932) containing essentially fatty acid-free albumin (final
concentration 2.5% w/v), the gas phase was O2/CO2 (95:5) and the temperature
was 37.5° C. The total incubation volume containing 65-95mg wet wt. of cells
and added substrates (5mM lactate, 5mM pyruvate, 5mM alanine, 5mM
glutamine, 5mM dihydroxyacetone, 5mM butyrate and 2mM [1-14C]oleate;
40µci/ml) was 4 ml. Incubations were stopped by addition of 0.4ml of 20% (w/v)
HClO4 , the mixture was centrifuged to remove denatured protein and the
supernatant was neutralized with KOH. The precipitate of KClO4 was removed
by centrifugation.
All incubations with [1-14C]Oleate were performed in duplicate in KrebsHenseleit saline (Krebs & Henseleit, 1932) containing dialysed albumin (fatty
acid poor, final concentration 2.5%, w/v). One of each pair was incubated in a
25ml Erlenmeyer flask with a centre well and rubber seal. The incubation was
stopped by the injection of 0.4 ml of 20 % (w/v) HClO4 with a long-needled
syringe (Whitelaw & Williamson, 1977). Sodium hydroxide (NaOH; 0.3 ml) was
injected into the centre well and metabolic CO2 collected by shaking the flasks
for a further 2 h at room temperature. The NaOH was then removed for
measurements of radioactivity and the deproteinised incubation medium was
treated as described above.
The reaction in the duplicate flask was stopped by centrifugation for 1
min at 5000 g to separate cells from the medium. Chloroform/methanol [2:1 (v/v);
20 vol.] was added to the cell pellet
and the lipids were extracted as described by Folch et al
(1957). The
radioactivity in the chloroform phase was used as a measure of the [1-14C]oleate
converted into esterified fats. The disappearance of radioactivity which was
extracted by chloroform from the incubation medium (minus pellet) was used to
calculate the oleate uptake. This was determined as follows: potassium phosphate
buffer (0.1 M) at pH 7.0 was added to the medium (1.5 ml/0.5 ml). The mixture
was extracted with chloroform (6 ml) by shaking for 90 sec. The chloroform and
aqueous phases were separated by centrifugation, the upper phase was removed
and the organic phase washed with 1 ml of potassium phosphate buffer, pH 7.0.
The radioactivity in a sample of the chloroform phase was measured. This method
relies on the fact that esterified lipids are not released in appreciable amounts to
the incubation medium, as shown by Whitelaw & Williamson (1976).
The radioactivity in ketone bodies was estimated by measuring the
radioactivity in acid-soluble compounds in the supernatant.
Calculation of rates:
The time-courses of glucose synthesis from the gluconeogenic precursors
and of ketone body formation from long- and short-chain fatty acids, in isolated
liver cells, were linear between 20 and 60 minutes, and were calculated as
follows:
The animals (starved 48h post-operation) were anaesthetized with an
intraperitoneal injection of pentobarbital (50mg/kg body wt). Each liver was
removed, minced and put into a pre-weighed ice-cooled 50 ml beaker containing
10ml of NaF sucrose (0.25 M sucrose; 50 mM NaF) buffer. More NaF sucrose
buffer was added to the beaker to a final volume/liver wt. of (10:1). Livers were
then homogenized using a glass homogenizer kept in ice. After homogenization
samples were centrifuged at 800 g (15 min), 16,000 g (20 min) and again at
16,000 g (20 min) to separate the microsomes from the cell wall, red cells and
cytosol during the first spin; from the mitochondria and the lysosomes during the
second spin; and from the remaining mitochondria and lysosomes during the third
spin. The supernatant of the third spin was carefully transferred to 12.5ml tubes
and ultracentrifuged for 1 h at 110,000 g to precipitate the microsomes. The
supernatant was then discarded and the microsomes were re-suspended in 20 mM
imidazole/chloride - 5 mM dithiothreitol buffer and assayed for HMGCoA
reductase activity.
HMGCoA Reductase
Buffer solutions required for the assay:
Buffer A
20mM Imidazole /Chloride
(pH 7.4)
5mM Dithiothreitol
Buffer B
350mM Potassium phosphate buffer
40mM EDTA
20mM Dithiothreitol
Cofactors
NADP+
10.5mg in 0.5ml H2O
Glucose-6-phosphate
Glucose-6-phosphate dehydrogenase
33.5mg in 0.5ml H2O
19U (50µl)+0.45ml H2O
These were mixed together. Buffer B (1.38ml) and H2O (0.38ml) were
added -
The cofactor mixture
Hydroxymethylglutaryl-CoA
reductase
activity
was
measured
as
described by Brown et al (1979). Microsomes, prepared as described previously,
were suspended in 20mM imidazole/chloride - 5 mM dithiothreitol (Buffer A)
and incubated in duplicate in the presence or absence of alkaline phosphatase at
37°C for 30 min , so that the total and the expressed amount of enzyme activity
could be measured. Before incubation portions (10 µl) of the homogenate were
taken in duplicate to measure the protein content. Three control incubations each
containing buffer A instead of microsomal homogenate. To the pre-incubated
microsomes the cofactor mixture was added and a solution of (R,S) [314C]HMG-CoA (80,000 dpm; 6.4 nmol; 8 µl) in KH2PO4 (0.1 M) was added to
each tube giving a final (R,S)-HMG-CoA concentration of 145 µM. Each tube
was capped and placed in a water bath at 37°C. The rack in which the tubes were
placed was shaken at 70 oscillations per min. Afer 20 min each tube was removed
from the water bath and the reaction stopped by the immediate addition of HCl
(12 M, 5 µl). An internal standard, consisting of a solution in water of (R,S)[23H ]mevalonolactone (5 µl - 10,986 dpm), was added to each tube. The
mevalonic acid was lactonised by incubating the tubes at 37°C for 30 min. At this
stage , if necessary , the samples could be stored for 1-3 days at -20°C.
Thin Layer Chromatography of Mevalonolactone
To precipitate protein , the tubes were centrifuged at 1000 g for 10 min.
Portions (40µl) of the supernatant of each sample were loaded into preadsorbent
strips of a multiple channel, glass-backed, Whatman LK5D silica gel TLC plate
(20 X 20 cm; 0.8 cm wide channels). The plates were thoroughly dried for 4 h in
a TLC tank
containing activated silica gel (coarse mesh). They were then
developed with ethylacetate/acetone (4:1,v/v) until the solvent, had reached 1 cm
from the top of the plate. The TLC tank contained 75 ml of the solvent, was lined
with chromatography paper and had been pre-equilibrated for at least 30 min. The
plates were dried and the mevalonolactone-containing band was located by
spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v) and
visualisation under ultra-violet light. This area of the plate was scraped from the
plates with a razor blade and transferred into scintillation glass vials. Ethanol (1
ml) was added, followed by 10 ml of a toluene-based scintillation cocktail
(PPO/POPOP/Toluene ; w/w/v ; 0.3 : 0.01 : 100). The 3H and 14C contents of the
sterol fraction were determined by scintillation counting for 30 min per sample.
Microsomal Protein Determination
The method of Lowry et al (1951) was used, as modified to assay protein
in the presence of sulphydryl reagents, by Geiger and Bessman (1972). The
standard curve was constructed using 0-150 µg of bovine serum albumin. After
the microsomes were re-suspended in 20mM imidazole/chloride - 5 mM
thiothreitol (Buffer A) and were ready for the incubation, a portion of the
suspension from each sample (10µl) was taken in duplicate into 10 ml tubes to
measure protein. The dithiothreitol was oxidised by heating in the presence of
H2O2.
Reagents:
A. 1 % CuSO4 5 H2O
B.
C.
5.4% NaK Tartrate
10% Na2CO3 ; 0.5 M NaOH
Copper Reagent:
1 ml of reagent A was mixed with 1 ml of reagent B
1 ml of this new mix (A+B) was added to 10 ml of reagent C
Phenol Reagent:
The phenol reagent was diluted with distilled water (v/v; 1:10)
Distilled water was added to the standards and the protein-containing
tubes up to a final volume per sample of 0.75 ml. The tubes were mixed and 0.75
ml of the copper reagent added; 15 µl of 3% H2O2 were also added to each tube
and they were mixed again. The tubes were transferred to a water bath at 60°C
for 10 min. Phenol reagent (1.5 ml) was added to each sample and standard ,
mixed and each tube transferred again to the water bath at 60°C for another 10
min. Tubes were cooled and each standard and each sample was transferred to the
same glass cuvette to be read in the spectrophotometer at 750 nM. The standard
curve was then constructed and the concentration of protein in the samples
calculated based on this standard curve.
Calculation of enzyme activity
The 14C dpm content of each sample was corrected for the recovery of [23H]mevalonolactone and by subtracting the average 14C dpm value obtained
from the control incubations (usually about 25 dpm). The corrected amount of
14C dpm was then converted to pmol of mevalonolactone (the specific
radioactivity of the substrate used was 12.5 dpm/pmol).
Therefore, the following equation was used to calculate enzyme activity:
HMG-CoA reductase activity was expressed as pmol of mevalonolactone
produced per min per mg of microsomal protein and was the average of duplicate
assays.
Determination of lipoprotein lipase activity
Lipoprotein lipase is an extracellular enzyme that is physiologically active
at luminal surface of the capillary endothelia of most extrahepatic tissues (for
review see; Cryer, 1981). It is responsible for the hydrolysis of plasma lipoprotein
triacylglycerol and, therefore, controls the rate limiting step in the removal of
triacylglycerol fatty acids from the circulation. The method used in this work to
measure lipoprotein lipase acitivity was essentially that described by Oller do
Nascimento & Williamson (1986). This involved the use of radioactive
triacylglycerol ([3H]-triolein), labelled in the fatty acid moiety, which is then
incorporated into a triacylglycerol emulsion from which the release of radioactive
fatty acid is measured, indicating the activity of lipoprotein lipase. The results
were expressed as nmol of free fatty acid released per min per mg of adipose
tissue.
Measurements of 3H and 14C radioactivity by liquid scintillation counting
were performed using an LS 9800 Liquid Scintillation System (Beckman
Instruments Inc., Irvine, CA, USA).
The sample to be measured was dissolved in a toluene-based scintillation
fluid (10 ml). Depending on the specific scintillation cocktail or radionuclide use,
distilled water and or ethanol was added to the vials. The vials were fitted with a
screw cap and when all the contents (sample, scintillation fluid and additions ;
e.g. water, ethanol) and were counted as described by Williamson et al (1975).
The counting procedure is based on the fact that ionising ß-particles
produced by the decay of 3H or 14C atoms lose some of their kinetic energy to
the scintillant. This energy is converted into photons. Initially the kinetic energy
is absorbed by the toluene and is subsequently transferred to the scintillant
present in the scintillation fluid. The photon emissions are detected as voltage
pulses, proportional in magnitude to the energy of the ß-particles, by two
photomultiplier tubes. Only coincident pulses are counted by the machine to
eliminate random events.
The overall yield for a particular radionuclide will be reduced by several
factors, which are termed quenching. The level of quenching was monitored and
the counting energy channels optimised for each sample by an automatic quench
compensation method. When in the dual-label counting mode (3H-14C), the
machine was calibrated with a series of differently quenched samples containing
either 3H or 14C of known dpm. The energy channels were set such that there
was no spillover from 3H into 14C. The quenching data and channel setting
during either single or dual-lable counting, were stored in the instrument as a 'user
counting program'. A microprocessor built into the machine automatically
calculated the 3H and/or 14C dpm using this program.
Experimental results were expressed as the mean ± the standard error of
the mean (S.E.M.) accompanied by the number of observations (n). The
representation used for mean values, and the mathematical definition of S.E.M.
are shown5 below:
Results were analysed using the Student t-test.
CHAPTER 4
Time course of changes in rates of gluconeogenesis in isolated
hepatocytes during induction of sepsis
Introduction
Carbohydrate metabolism is substantially affected by sepsis. Glycogen
reserves are depleted in early sepsis, during the fuel mobilization characteristic of the
ebb phase. During the flow phase, when sepsis is usually accompanied by diminished
or virtually no carbohydrate intake, there is increased demand for glucose, especially
by glucose-dependent tissues including the brain, nerve cells, red blood cells, kidney
medulla, and the cells involved in the infectious-inflamatory reaction and the repair
process (Frayn KN, 1986). This requirement for glucose is met by the synthesis of
glucose de novo - gluconeogenesis (Fig. 4.1.). The catabolism of glucose in these
glycolytic tissues forms a large amount of lactate (Kelleher et al , 1982) which is taken
up by the liver as a prime precursor for glucose synthesis.
During sepsis, the liver uses other substrates to produce glucose such as
pyruvate, glycerol, alanine and other gluconeogenic amino acids. The contribution of
these precursors reported in the literature varies, e.g. alanine utilization for hepatic
gluconeogenesis, in the presence of sepsis, has been reported to range from 30 to
70% (Forse & Kinney, 1985).
The increased muscle proteolysis induced by sepsis (Long et al , 1977;
Hasselgren et al , 1986) and the reported accelerated metabolism of branched chain
amino acids with formation of alanine during sepsis (Biesel & Wannemacher, 1980)
provide a continuous supply of precursors for glucose
synthesis from muscle.
Glucagon and catecholamines stimulate gluconeogenesis by their ability to
activate adenylate cyclase, increasing the concentration of cyclic AMP in the
hepatocytes (Newsholme & Leech, 1983). Both hormones are reported to be increased
during sepsis (Cerra et al , 1980; Watters et al , 1986). Insulin, however, is known to
antagonize adrenaline or glucagon action, by lowering the concentration of cyclic AMP
within the liver cells, inhibiting gluconeogenesis (Newsholme & Leech, 1983). Higher
concentrations of plasma insulin were found in caecal-ligated and punctured rats (Fig.
3.5. - Chapter 3). This has also been reported in other forms of sepsis (Biesel &
Wannemacher, 1980; Neufeld et al , 1980 and 1982; Clemens et al , 1984; Shaw et al ,
1985).
Thus during sepsis some conditions favour increased glucose synthesis,
namely, increased availability of substrates (Figs. 3.2.; 3.3.; 3.4 - Chapter 3) and
elevated concentrations of gluconeogenic hormones. On the other hand, the presence
of increased concentrations of plasma insulin may inhibit gluconeogenesis. It is
therefore perhaps not surprising that there is controversy regarding the effects of
sepsis on hepatic glucose metabolism. In some studies gluconeogenesis has been
reported to be increased (Wannemacher et al , 1980; Lang et al , 1984; Kelleher et al ,
1982; Shaw et al , 1985) while in others it is decreased (Wannemacher et al , 1976;
Wilmore DW, 1977;Guillem et al , 1982; Clemens et al , 1984). Such widely differing
results are presumably due to several factors such as the source and type of sepsis,
the nutritional and hormonal status of the animal and the stage of the infection.
To investigate changes with time in the rate of gluconeogenesis, hepatocytes
were isolated from caecal-ligated and punctured and sham-operated rats, and glucose
synthesis from various gluconeogenic precursors measured in vitro.
Design of the study
Rats, after being made septic by caecal-ligation and puncture or subjected to shamoperation, were starved for 12, 24 or 48 h before isolation of hepatocytes (Fig 4.2.).
Fig 4.2.
OP
12 h
24 h
48 h
Hepatocyte isolation
Rats
Incubation
1. no substrate
2. lactate
3. pyruvate
20'
40'
4. alanine
5. glutamine
6. dihydroxyacetone
60'
For details of the technique of hepatocyte isolation and incubation procedures see
Chapter 2. Cells from caecal-ligated and punctured rats and from sham-operated rats
were incubated with various gluconeogenic precursors at 12, 24 and 48 h after the
operation (Fig. 4.2.) and rates of glucose synthesis were measured.
Results
The basal rates of glucose formation by hepatocytes from septic and shamoperated rats were low and similar at 12, 24 and 48 h after the operation (Fig. 4.3)
Fig. 4.3.
Rate of gluconeogenesis - no substrate
(5)
µmol/g wet wt. cells/min
0.20
(5)
(5)
(8)
(5)
Sham
Sham-op
Septic
(6)
0.10
X + S.E.M.
( ) nº of observations
0.00
12
1
24
2
48
3
Time after the operation (h)
The addition of various gluconeogenic precursors, namely lactate, alanine,
pyruvate, glutamine and dihydroxyacetone, at the final concentration of 5 mM, resulted
in increases of 3- to 4-fold in the rates of glucose synthesis as compared to basal
rates. However, hepatocytes from caecal-ligated and punctured rats produced glucose
at significantly lower rates when compared to those of sham-operated rats.
When lactate was the substrate the rate of glucose synthesis, at 24 and 48 h
after the operation, was significantly lower when compared to those of sham-operated
rats (Fig. 4.4.).
Fig. 4.4.
Rate of gluconeogenesis from lactate [5mM]
(5)
0.8
**
(5)
0.6
(5)
0.4
(8)
*
(6)
(5)
Sham-op
Sham
Septic
* P < 0.05
** P < 0.01
0.2
X + S.E.M.
0.0
12
1
24
2
48
3
Clemens et al (1984), using a caecal-ligation and double puncture model, also found
decreased production of glucose from lactate [5mM], in perfused livers from septic rats
stimulated with phenylephrine, as early as at 6 h after the operation.
When hepatocytes were incubated with lactate, at various concentrations,
rates of glucose synthesis were progressively lower in isolated liver cells from caecalligated rats (n=4) as the concentration of lactate in the incubation medium increased,
when compared to those of hepatocytes from sham-operated rats (n=3) at 48 h after
the operation (Fig. 4.5). This depression in glucose synthesis in isolated liver cells from
septic rats could be overcome when hepatocytes were incubated with lactate at similar
concentration to those found in the blood in vivo (septic [2.5mM]; sham-operated
[1.5mM] - see Fig. 3.2. - Chapter 3). Under these conditions the rate of glucose
production was approximately the same (0.51 µmol/g wet wt. cells/min - Fig. 4.5.).
This suggests that provided the availability of gluconeogenic
precursors is increased by sepsis, as indicated by the increased concentrations of
lactate, pyruvate and alanine in the blood and in the liver of caecal-ligated and
punctured rats (see Chapter 3), gluconeogenesis in vivo may be maintained during
sepsis.
Fig. 4.5.
Rates of gluconeogenesis from lactate
0.8
0.7
0.6
Sham-op
Septic
0.5
0.4
0.3
X ± S.E.M.
48 h after the operation
0.2
0.1
[0]
[0.625]
[[1.25]
[2.5]
[5]
[10]
Incubation concentration [mM]
Gluconeogenic rates in hepatocytes from septic rats were significantly lower
than those of liver cells isolated from sham-operated rats (at 24 h and 48 h) when
alanine [5mM] was the substrate (Fig. 4.6.).
Fig. 4.6.
Rate of glucose synthesis from alanine [5mM]
0.50
0.40
(5)
*
(5)
(5)
**
(6)
(5)
0.30
Sham-op
Septic
(8)
* P < 0.05
0.20
** P < 0.01
0.10
X + S.E.M.
0.00
12
1
24
2
48
3
Perfused livers from septic rats, subjected to caecal-ligation and double puncture,
produced glucose from alanine [10mM] at significantly lower rates than perfused livers
from sham-operated rats at 18 h after the operations (Guillem et al , 1982). However,
rates of gluconeogenesis in vivo
from [1-14C]alanine increased in rats with
pneumoccocal infection as compared to fasted control rats (Wannemacher et al ,
1980).
When pyruvate was the substrate, isolated liver cells from caecal-ligated and
punctured rats formed glucose at significantly lower rates at all time points studied (12
h, 24 h and 48 h - Fig. 4.7.).
Fig. 4.7.
Rate of gluconeogenesis from pyruvate [5mM]
0.70
(5)
0.60
(8)
0.50
(5)
*
(5)
**
(5)
0.40
Sham-op
Septic
**
(6)
* P < 0.05
0.30
** P < 0.01
0.20
X + S.E.M.
0.10
0.00
12
1
24
2
48
3
Hepatocytes from rats with pneumoccocal infection
synthesized glucose
from pyruvate [10mM] at lower rates than hepatocytes from fasted control rats 40 h
after the injection of bacteria; at 24 h after bacterial injection rates of gluconeogenesis
were similar to those of controls (Wannemacher et al , 1980).
Hepatocytes from septic rats produced glucose from glutamine at
significantly lower rates as compared to those of isolated liver cells (24 h and 48 h)
from sham-operated rats (Fig. 4.8.).
Fig. 4.8.
Rate of gluconeogenesis from glutamine [5mM]
(8)
0.50
(5)
0.40
(5)
0.30
*
*
(5)
(5)
Sham-op
Septic
(6)
0.20
* P < 0.01
0.10
X + S.E.M.
0.00
12
1
24
2
48
3
The reason for this difference in the rate of glucose formation from glutamine, between
the two groups of cells, appeared to be due to the fact that the rates of glucose
synthesis in hepatocytes from sham-operated rats increased with time while those of
liver cells from septic rats remained unchanged with the progression of sepsis.
Sepsis had no effect on the production of glucose from dihydroxyacetone at
12 and 24 h after the operations. However, rates of glucose synthesis in isolated liver
cells from septic rats were significantly lower at 48 h after the operation as compared to
those of hepatocytes from sham-operated animals (Fig. 4.9.).
Fig. 4.9.
Rate of gluconeogenesis from dihydroxyacetone [5mM]
1.2
1.0
(8)
(5)
(5)
(5)
(5)
*
(6)
0.8
Sham-op
Septic
0.6
* P < 0.01
0.4
X + S.E.M.
0.2
0.0
12
1
24
2
48
3
Discussion
Despite causing no appreciable change in the basal rate of production of
glucose, at the time points studied, gluconeogenesis was progressively and
significantly impaired in hepatocytes isolated from septic rats in the presence of added
substrates. This inhibition of glucose synthesis in vitro appears to affect all substrates
tested at 48 h after the operation, when rates of glucose formation were approximately
35% lower in hepatocytes from septic rats, despite the fact the gluconeogenic
precursors used enter into the gluconeogenic pathway at different levels (Fig. 4.1.).
However, if hepatocytes were incubated with lactate at similar concentrations
to those found in the blood in vivo (Fig. 3.2. - Chapter 3; Fig. 4.5.), the rate of glucose
production was about the same suggesting gluconeogenesis in vivo
may
be
maintained in sepsis by increasing the
availability of hepatic glucose precursors (Figs. 3.2.; 3.3.; 3.4. - Chapter 3). A similar
conclusion has been reached by Spitzer et al (1985) studying hepatocytes isolated
from rats treated with continuous infusion of endotoxin.
Injection of endotoxin into mice decreases activities of the gluconeogenic
enzymes (Fig. 4.1.), glucose 6-phosphatase, fructose 1-6-biphosphatase and
phosphoenolpyruvate carboxykinase (McCallum & Berry, 1972; Ripe & Berry, 1972).
The endotoxin treatment appears
to inhibit the glucocorticoid induction of
phosphoenolpyruvate carboxykinase at a late post-receptor induction step (Shackleford
et al , 1986). Very recent evidence suggests stimulation of the glycolytic enzyme, 6phosphofructokinase, as the primary site of inhibition in vitro of gluconeogenesis by
endotoxin injection into rats (Knowles et al , 1987); it would therefore be predictable
that the concentrations of intermediates preceding the fructose bisphosphatase/PKF-1
cycle should be increased. Against this view is the fact that, in the present studies,
hepatic concentrations of glucose-6-phosphate were significantly elevated in septic rats
at 24 and 48 h after the operation (Figs. 3.15 & 3.16 - Chapter 3). It is known that
addition of endotoxin does not inhibit hepatic gluconeogenesis in vitro (Filkins &
Cornell, 1974), which suggests that its effects in vivo are indirect. Recent evidence
supports the view that endotoxin exerts most of its metabolic effects via the action of
endogenous mediators produced by cells of haemotopoietic origin (Tracey et al , 1986;
Beutler & Cerami, 1987). There is also evidence that pneumoccocal infection
decreases the hepatic activity of glucose 6-phosphatase by 50% (Cannonico et al ,
1977). In the present studies, accumulation of hepatic gluconeogenic intermediates up
to glucose-6-phosphate, with no change in glucose concentration in the livers of septic
rats (Table 3.1.; Fig. 3.15. - Chapter 3), suggests an inhibition of gluconeogenic flux at
the level of glucose 6-phosphatase. The unchanged concentration of hepatic or blood
glucose presumably reflecting a balance between decreased peripheral utilization and
hepatic production.
If sepsis causes similar changes in enzyme concentrations in the rat this
would explain the decreased capacity for hepatic gluconeogenesis in vitro . The
decrease in hepatic acetyl-CoA concentration in vivo (Fig. 3.17. - Chapter 3), which is
an obligatory activator of pyruvate carboxylase (Fig. 4.1.; Scrutton & Griffiths, 1981),
may play a role in diminishing flux through this enzyme, therefore, also limiting
gluconeogenesis during sepsis.
The absence of hypoglycaemia in the septic rats (Fig. 3.1.- Chapter 3)
suggests that this impairment of gluconeogenesis may be overcome in vivo
by
increased concentration of precursors, mainly lactate and alanine, which are supplied
in increased amount from muscle in sepsis (Biesel & Wannemacher, 1980; Hasselgren
et al , 1984).
CHAPTER 5
Time course of changes in the metabolism of [1-14C]oleate and the rate of ketogenesis
in isolated liver cells after induction of sepsis
Introduction
Sepsis in man and experimental animals causes a number of changes in lipid
metabolism (Blackburn, 1977; Beisel & Wannemacher, 1980; Neufeld et al , 1982). These
include hypertriacylglycerolaemia (Blackburn, 1977; Beisel
& Wannemacher, 1980),
decreased blood ketone bodies (Neufeld et al , 1976) and changes in plasma free fatty acid
concentrations and lipoprotein lipase activity (Robin et al , 1981; Lanza-Jacoby et al ,
1982). It is therefore, important to obtain information on the effects of sepsis on the hepatic
metabolism of fatty acids during starvation.
During starvation plasma non-esterified fatty acids are derived exclusively
from the hydrolysis of triacylglycerol within the adipose tissue and the rate of mobilization
of fatty acids from this tissue controls their concentration in the plasma. The liver has the
capacity to remove a large proportion of non-esterified fatty acids from the circulation
(Newsholme & Start, 1973). The rate of hepatic uptake
appears to be regulated by the concentration of non-esterified fatty acids in plasma. Once
in the hepatocyte, these non-esterified fatty acids can be esterified to form triacylglycerol
or be partially oxidized to form ketone bodies, or be totally oxidized into the Krebs cycle
to form metabolic CO2 (Fig. 5.1.). Long-chain fatty acids have to be activated and
transported into the mitochondria for oxidation via the carnitine acyl-CoA transferase
system, whereas short-chain fatty acids are not be esterified and do not require the acylCoA transport system to enter the mitochondria for ß-oxidation (Fig. 5.1.; for review see
McGarry & Foster, 1980).
Fig. 5.1.
Changes in plasma non-esterified fatty acids and blood and hepatic ketone bodies
concentrations during sepsis
While plasma non-esterified fatty acid concentrations have been reported to be
variable, esterification by the liver has been reported to be increased by sepsis (Beisel &
Wannemacher, 1980). The
increased production of triacylglycerol by the liver together with diminished activity of
lipoprotein lipase in peripheral tissues have been proposed as the mechanism responsible
for hypertriacylglycerolaemia during sepsis (Robin et al , 1981; Lanza-Jacoby et al , 1982).
During the fed state, the concentration of ketone bodies (acetoacetate and 3hydroxybutyrate) in the liver and blood is very low (Williamson et al , 1967). During the
starved state, large quantities of ketone bodies are produced by the liver and are
transported via the blood to peripheral tissues where they are used as important alternative
substrates. The acetyl-CoA formed in the metabolism of ketone bodies can either be
oxidized in the Krebs cycle to
provide energy or in particular tissues, such as the
developing brain or lactating mammary gland, it can be used as precursor for lipid
synthesis (Williamson & Whitelaw, 1978; Williamson, 1981).
However, if starvation is accompanied by sepsis, blood and hepatic concentrations
of ketone bodies decrease to values similar to those of the fed state (Neufeld et al, 1976
and 1977). Possible reasons for this failure to develop hyperketonaemia, in the starved
septic state, are increased utilization of ketone bodies or decreased ketone body formation.
The latter may be the result of poor perfusion of adipose tissue leading to decreased
mobilization of non-esterified fatty acids, and/or decreased hepatic blood flow
(Williamson, 1981). Increased utilization in peripheral tissues does not appear to be the
cause because infused 3-hydroxybutyrate reaches similar blood
concentrations, in infected as well as in fasted control sheep (Radcliffe et al , 1981).
Wannemacher et al (1979), studying pneumococcal sepsis in rats, proposed that the
failure of septic rats to become ketonaemic during starvation was the result of reduced
ketogenic capacity of the liver and , possibly, a decrease in the hepatic supply of fatty
acids.
In order to obtain more information and understand the mechanisms reponsible for
the failure to develop during sepsis the hyperketonaemia associated with starvation , rates
of ketogenesis from long- and short-chain fatty acids in isolated hepatocytes, from caecalligated and punctured rats as compared to sham-operated animals, were measured. The
metabolism of [1-14C]oleate, namely the rate of uptake and distribution into the pathways
of esterification, ketogenesis and 14CO2 formation, by isolated liver cells from septic and
sham-operated rats, was also studied.
Rats were subjected to caecal-ligation and puncture or sham-operation and
starved for 12, 24 or 48 h when they were studied (Fig 5.2.).
Fig 5.2.
OP
12 h
24 h
48 h
Rats
Incubation
1. no substrate
2. butyrate
3. oleate
20'
40'
60'
For details of the technique of liver cell isolation and incubation procedures see Chapter 2.
Isolated liver cells from a group of rats, studied at 48 h after the operation, were incubated
with [1-14C]oleate. The uptake and the conversion of [1-14C]oleate into its metabolic
products were studied.
Results
Time course of rates of ketogenesis from long- and short-chain fatty acids in isolated
hepatocytes
There were no appreciable changes in the rate of ketone body formation at
any of the time points studied in the absence of added substrate (Fig. 5.3.).
Fig. 5.3.
When hepatocytes from caecal-ligated and punctured rats and sham-operated rats
were incubated with [5mM]butyrate, a short-chain fatty acid, there were no significant
differences in the rate of ketogenesis (Fig. 5.4.).
Fig. 5.4.
However, isolated liver cells from septic rats produced ketone bodies at a
significantly lower rate, at all time points studied, when the long-chain fatty acid
[2mM]oleate was the substrate (Fig. 5.5.).
Fig. 5.5.
Similar results were found in isolated perfused livers from rats with pneumoccocal
infection (Wannemacher et al , 1979).
Table 5.1. shows the uptake and conversion of [1-14C]oleate to its
metabolic products. The rate of ketone body formation from [1-14C]oleate was
significantly lower in septic rats as compared with sham-operated rats, due to a decreased
rate of D-3-hydroxybutyrate formation (Table 5.1.). This change in the [3hydroxybutyrate]/ [acetoacetate] ratio would be expected if ß-oxidation is depressed
(Williamson et al , 1967). The rate of removal of [1-14C]oleate from the medium (uptake)
by isolated liver cells from septic rats was 23% lower as compared with sham-operated
animals (Table 5.1. ; Fig. 5.6.).
Although there was no significant difference in the absolute amounts of [114C]oleate esterified by hepatocytes from septic rats (Table 5.1.), the percentage was
significantly higher (Fig. 5.6.) than that of sham-operated rats (45±2.8% versus 36±0.73%
; P < 0.05, n=6). The percentage of [1-14C]oleate converted to metabolic 14CO2 in
hepatocytes from septic rats was twice (Fig. 5.6.) that of sham-operated rats (3.5±0.5%
versus 1.8±0.02% ; P < 0.05, n=6) and may be a contributory factor to the decreased
ketogenesis as it reflects more acetyl-CoA entering the Krebs cycle in the hepatocytes from
septic rats. The rate of formation of acid-soluble (A-S) products, mainly ketone bodies,
was significantly lower in hepatocytes from septic rats (Table 5.1.; Fig. 5.6.) as was the
percentage of [1-14C]oleate converted to these products ( 40±4.5% versus 60±4.8% ; P
< 0.05, n=6).
Table 5.1.
Effects of sepsis on metabolism of [1-14C]oleate in isolated hepatocytes from 48 h starved
rats
_________________________________________________________________________
Results are mean values ± SEM with number of observations shown in parentheses.
Significance of difference by Student's t-test from the sham-operated rats: * P < 0.01 ; ** P < 0.001.
__________________________________________________________________
_______
Rate (µmol/g wet wt. cells/min)
_________________________________________________________
Sham-operated
Septic
Change (%)
_______________________________________________________________________
Acetoacetate
0.52±0.027 (6)
0.55±0.055 (6)
+ 6
3-Hydroxybutyrate
1.15±0.062 (6)
0.66±0.056 (6) **
- 57
Total ketone bodies
1.66±0.058 (6)
1.23±0.077 (6) **
- 26
Removal of [1-14]oleate
0.55±0.044 (6)
0.42±0.053 (6)
- 23
Esterification of [1-14C]oleate
[1-14C]oleate - 14CO2
0.20±0.004 (6)
0.19±0.012 (6)
- 5
0.010±0.001 (6)
0.015±0.002 (6)
+ 50
[1-14C]oleate - 14C acid
0.33±0.028 (6)
0.17±0.020 (6) *
- 48
soluble products
_______________________________________________________________________
Fig. 5.6.
Metabolism of [1 - C]oleate14
% [1-14C]oleate distribution
100
80
Esterification
A-S products
Metabolic CO 2
60
40
20
0
Sham-op
Discussion
Septic
The concentration of plasma non-esterified fatty acids has been reported to be
increased (Blackburn, 1977) or decreased (Neufeld et al , 1982) by sepsis. In the present
study the slight decrease (13% ; Fig. 3.7. - Chapter 3) was not significant. Nevertheless, in
vitro ketogenesis from the added long-chain fatty acid, oleate, was markedly depressed
(Fig. 5.5.) indicating that the septic liver has a decreased capacity to produce ketone
bodies. Others have shown that perfused livers from rats with pneumococcal infection
produce significantly less ketone bodies from oleate as compared to perfused livers from
fasted controls (Wannemacher et al , 1979).
When medium chain fatty acids, such as octanoic acid, are the substrate, isolated
perfused livers from infected rats produce as much ketone bodies as livers from starved
controls (Wannemacher et al , 1979). Similar results were found when butyrate was the
substrate in isolated liver cells from caecal-ligated and punctured rats, as compared to
hepatocytes from sham-operated rats (Fig. 5.4.)
The fact that ketogenesis remained unchanged, in isolated hepatocytes from septic
rats, when the short-chain fatty acid butyrate was the substrate, whereas it was significantly
lower when the long-chain fatty acid oleate was the substrate, suggests that a site of
inhibition of ketogenesis in the septic liver may be the entry of long-chain fatty acid into
the mitochondria via the carnitine acyl-CoA transferase system (McGarry & Foster , 1980).
Long-chain fatty acyl-CoA would, alternatively, go into the esterification pathway leading
to formation of triacylglycerols and phospholipids.
One physiologically important regulatory mechanism of the entry of long-chain
acyl-CoA into the mitochondria is the modulation of the flux through carnitine
palmitoyltransferase I by malonyl-CoA, an intermediate of the lipogenic pathway.
Although the precise mechanism of malonyl-CoA inhibition on carnitine acyltransferase I
in the rat liver mitochondria has not yet been fully established, available evidence indicates
a competitive type of inhibition against long-chain acyl-CoA substrates (McGarry &
Foster, 1980). During starvation, at low rates of lipogenesis, decreased malonyl-CoA
concentrations permit the diversion of long-chain fatty acid into oxidative pathways in the
mitochondria. However, despite the fact that donor rats are starved, hepatocytes from
septic rats divert more long-chain fatty acid to esterification (Fig. 5.6.) than to partial
oxidation in the mitochondria to yield ketone bodies (Fig 5.5.).
The rate of hepatic lipogenesis in vivo is significantly increased (Fig. 6.2. Chapter 6) in starved septic rats. Although lipogenesis is considered to be positively
correlated with cytosolic malonyl-CoA concentrations it must be emphasized that hepatic
carnitine acyl-CoA transferase I (CAT I) is less sensitive to inhibition by malonyl-CoA in
livers from starved rats (Cook et al , 1980).
Sepsis induced by injection of bacteria has been reported not to increase hepatic
malonyl-CoA (Wannemacher et al , 1979) or alternatively to increase this metabolite (Vary
et al , 1986). There is also evidence that the sensitivity of carnitine acyl transferase I to
malonyl-CoA can be rapidly increased by insulin (Gamble & Cook, 1985) and, therefore, it
may be pertinent that our septic rats had higher plasma insulin concentration (Fig. 3.5. Chapter 3). It must however be emphasised that lipogenesis has not been measured in the
present hepatocyte experiments.
A decreased [D-3-hydroxybutyrate]/[acetoacetate] ratio (Table 5.1.) together with
the lower concentrations of acetyl-CoA ( Fig. 3.17. - Chapter 3) in livers from septic rats
support the view of depressed ß-oxidation in sepsis, as a result of the diminished flux of
long-chain acyl-CoA into the mitochondria. An alternative reason for the decreased entry
of long-chain acyl-CoA into the mitochondria is that the high insulin concentrations may
suppress the putative role of glucagon to phosphorylate and increase the activity of
carnitine acyl transferase I. Recent experiments have suggested that glucagon can increase
the affinity of the enzyme complex (CPT1+CPT2) for palmitoyl-CoA and increase the
phosphorylation of the protein (Harano et al , 1985). Further work has suggested that CPT
activity might be regulated by both protein kinase A and by Ca++/calmodulin-dependent
protein kinase activities (Kojima et al , 1986).
The reason for the impairment of ketogenesis in livers from septic rats, therefore,
appears to be multifactorial and involves a possible decrease in hepatic uptake of nonesterified fatty acids, depression of the entry of long-chain acyl-CoA into the mitochondria
for ß-oxidation and lower hepatic concentration of acetyl-CoA. The increase in the
percentage of metabolic CO2 formation, by hepatocytes from septic rats, may be another
contributory factor for it indicates more acetyl-CoA entering the Krebs cycle for oxidation,
thus decreasing the availability of acetyl-CoA as the immediate cause for the decreased
ketogenesis.
This depression of the entry of long-chain acyl-CoA may be of clinical importance
in deciding what lipid emulsion to infuse, as part of nutrients required, in intravenous
nutrition. The use of recently available lipid emulsions, which are enriched with mediumchain fatty acids, might be theoretically beneficial for septic patients (Eckhart et al ,
1980). However reports on the use of these long-chain triacylglycerol (LCT)/mediumchain triacylglycerol (MCT) emulsions are controversial. Stein et al (1986) studied caecalligated rats receiving intravenous nutrition with either MCT/LCT or LCT emulsions as
part of or as the only non-protein calorie source. Rats fed with
MCT/LCT mixture alone, or in association with glucose, showed
significantly lower survival rates as compared to rats fed with other
nutrient mixtures. In contrast, Dennison et al (1986), in a randomized crossover trial,
showed a significant increase in blood ketone body concentration accompanied by a
decrease in plasma tricylglycerol concentration, in patients parenterally fed with half of
their non-nitrogen calories as an MCT/LCT (1:1) 10% lipid emulsion, as compared to the
concentrations measured when the same patients were receiving a 10% lipid emulsion
containing only LCT. The increase in ketone body concentration suggests increased
hepatic utilization, whereas the decrease in plasma triacylglycerol suggests enhanced
clearance with MCT. While receiving MCT, patients also had a better nitrogen retention.
Therefore, if the depression in the entry of long-chain acyl-CoA in the liver of the
septic rat is also present in the liver of septic patients, MCT containing lipid emulsions
may be theoretically regarded as a better caloric source during sepsis. Nevertheless further
investigation, in the form of randomized control studies involving septic patients requiring
intravenous feeding is needed.
CHAPTER 5
Time course of changes in the metabolism of [1-14C]oleate and
ketogenesis in isolated liver cells after induction of sepsis
Introduction
Sepsis in man and experimental animals causes a number of changes in
lipid metabolism (Blackburn, 1977; Beisel & Wannemacher, 1980; Neufeld et al , 1982).
These include hypertriacylglycerolaemia (Blackburn, 1977; Beisel & Wannemacher,
1980), decreased blood ketone bodies (Neufeld et al , 1976) and changes in plasma
free fatty acids concentration and lipoprotein lipase activity (Robin et al , 1981; LanzaJacoby et al , 1982). Therefore, a better understanding of the hepatic metabolism of
long- and short-chain fatty acids during starvation in the presence or absence of sepsis
is necessary.
During starvation plasma non-esterified fatty acids are derived exclusively
from the hydrolysis of triacylglycerol within the adipose tissue and the rate of
mobilization of fatty acids from this tissue controls their concentration in the plasma.
The liver has the capacity to remove a large proportion of non-esterified fatty acids
from the circulation (Newsholme & Start, 1973). The rate of hepatic uptake appears to
be regulated by the concentration of non-esterified fatty acids in plasma. Once in the
hepatocyte, these non-esterified fatty acids can be esterified to form triacylglycerol or
be partially oxidized to form ketone bodies, or be totally oxidized into the Krebs cycle to
form metabolic CO2 (Fig. 5.1.). Long-chain fatty acids have to be activated and
transported into the mitochondria for oxidation via the carnitine acyl-CoA transferase
system, whereas short-chain fatty acids can not be esterified and do not require the
acyl-CoA transport system to enter the mitochondria and suffer ∫-oxidation (Fig. 5.1.; for
review see McGarry & Foster, 1980).
Fig. 5.1.
Simplified scheme of the hepatic metabolism of long- and short-chain fatty acids
Long-chain fatty acids
Short-chain fatty acids
Long-chain fatty acyl-CoA
carnitine
Esterified
CAT I
products
Lactate
CAT II
Pyruvate
VLDL
carnitine
Fatty acyl-CoA
Oxaloacetate
Short-chain
fatty
acyl-CoA
Acetyl-CoA
Acetoacetyl-CoA
HMG-CoA
CO
2
CO
2
Acetoacetate
ß-Hydroxybutyrate
Changes in plasma non-esterified fatty acids and blood and hepatic ketone bodies
concentrations during sepsis
While plasma non-
esterified fatty acid concentrations have been reported to be variable, esterification by
the liver has been reported to be increased by sepsis (Beisel & Wannemacher, 1980).
The increased production of triacylglycerol by the liver together with diminished activity
of lipoprotein lipase in peripheral tissues have been proposed as the mechanism
responsible for hypertriacyglycerolaemia during sepsis (Robin et al, 1981; LanzaJacoby et al , 1982).
During the fed state, the concentration of ketone bodies (acetoacetate and
3-hydroxybutyrate) in the liver and blood is very low (Williamson et al , 1967). During
the starved state, when the supply of carbohydrate is limited, large quantities of ketone
bodies are produced by the liver and are transported via the blood to peripheral tissues
where they are used as important alternative substrates. The acetyl-CoA formed in the
metabolism of ketone bodies can either be oxidized in the Krebs cycle to provide
energy or in particular tissues, such as, the developing brain or lactating mammary
gland, it can be used as precursor for lipid synthesis (Williamson & Whitelaw, 1978;
Williamson, 1981).
However, if starvation is accompanied by sepsis, blood and hepatic
concentrations of ketone bodies decrease to values not different from those of the fed
state (Neufeld et al, 1976 and 1977). Possible reasons for this failure to develop
hyperketonaemia, in the starved septic state, are increased utilization of ketone bodies
or decreased ketone body formation. The latter may be the result of poor perfusion of
adipose tissue leading to decreased mobilization of non-esterified fatty acids, and/or
decreased hepatic blood flow (Williamson DH, 1981). Increased utilization in peripheral
tissues does not appear to be the cause because infused 3-hydroxybutyrate was
oxidised to similar amounts of acetoacetate, and both ketone bodies were equally
cleared, in infected as well as in fasted control sheep (Radcliffe et al , 1981).
Wannemacher et al (1979), studying pneumococcal sepsis in rats, proposed that the
failure of septic rats to become ketonemic during starvation was the result of reduced
ketogenic capacity of the liver and , possibly, a decrease in the hepatic supply of fatty
acids.
In order to obtain more information and understand the mechanisms
reponsible for the failure to develop during sepsis the hyperketonaemia associated with
starvation , rates of ketogenesis from long- and short-chain fatty acids in isolated
hepatocytes, from caecal-ligated and punctured rats as compared to sham-operated
animals, were measured. The metabolism of [1-14C]oleate, namely the rate of uptake
and distribution into esterification, ketogenesis and 14CO2 formation, by isolated liver
cells from septic and sham-operated rats, was also studied.
Rats were subjected to caecal-ligation and puncture or sham-operation
and starved for 12, 24 or 48 h when they were studied (Fig 5.2.).
Fig 5.2.
OP
12 h
24 h
48 h
Rats
Incubation
1. no substrate
2. butyrate
3. oleate
20'
40'
60'
For details of the technique of liver cell isolation and incubation procedures see
Chapter 2.
Isolated liver cells from a group of rats, studied at 48 h after the operation,
were incubated with [1-14C]oleate. The uptake and the conversion of [1-14C]oleate
into its metabolic products were studied.
Results
Time course of rates of ketogenesis from long- and short-chain fatty acids in isolated
hepatocytes
There were no appreciable changes in the rate of ketone body formation at
any of the time points studied in
the absence of added substrate (Fig. 5.3.).
Fig. 5.3.
Rate of ketone body formation - no added substrate
0.5
(5)
0.4
(5)
(3)
(3)
(5)
(5)
0.3
Sham-op
Septic
0.2
X + S.E.M.
0.1
0.0
12
1
24
2
48
3
When hepatocytes from caecal-ligated and punctured rats and shamoperated rats were offered [5mM]butyrate, a short-chain fatty acid, as the substrate
there were no significant differences in the rate of ketogenesis (Fig. 5.4.).
Fig. 5.4.
Rate of ketogenesis from butyrate [5mM]
2
(5)
(5)
(5)
(3)
(3)
(5)
Sham-op
Septic
1
X + S.E.M.
0
12
1
24
2
48
3
However, isolated liver cells from septic rats produced ketone bodies at a
significantly lower rate, at all time points studied, when the long-chain fatty acid
[2mM]oleate was the substrate (Fig. 5.5.).
Fig. 5.5.
Rate of ketogenesis from oleate [2mM]
(7)
1.50
*
(8)
(3)
(4)
1.00
Sham-op
Septic
**
(4)
*
(5)
* P < 0.05
** P < 0.01
0.50
X + S.E.M.
0.00
12
1
24
2
48
3
Similar results were found in isolated perfused livers from rats with pneumoccocal
infection (Wannemacher et al , 1979).
Table 5.1. shows the uptake and conversion of [1-14C]oleate to its
metabolic products.
The rate of ketone body formation from [1-14C]oleate was significantly
lower in septic rats as compared with sham-operated rats, due to a decreased rate of
D-3-hydroxybutyrate
formation
(Table
5.1.).
This
change
in
the
[3-
hydroxybutyrate]/[acetoacetate] ratio would be expected if ∫-oxidation is depressed
(Williamson et al, 1967). The rate of removal of [1-14C]oleate from the medium
(uptake) by isolated liver cells from septic rats was 23% lower as compared with shamoperated animals (Table 5.1. ; Fig. 5.6.).
Although there was no significant difference in the absolute amounts of [114C]oleate esterified by hepatocytes from septic rats (Table 5.1.), the percentage was
significantly higher (Fig. 5.6.) than that of sham-operated rats (45±2.8% versus
36±0.73% ; P < 0.05, n=6). The percentage of [1-14C]oleate converted to metabolic
14CO in hepatocytes from septic rats was twice (Fig. 5.6.) that of sham-operated rats
2
(3.5±0.5% versus 1.8±0.02% ; P < 0.05, n=6) and may be a contributory factor to the
decreased ketogenesis as it reflects more acetyl-CoA entering the Krebs cycle in the
hepatocytes from septic rats.
Table 5.1.
Effects of sepsis on metabolism of [1-14C]oleate in isolated hepatocytes from 48
h starved rats
_______________________________________________________________________
Results are mean values ± SEM with number of observations shown in parentheses.
Significance of difference by Student's t-test from the sham-operated rats: * P < 0.01 ;
** P < 0.001.
_________________________________________________________
Rate (µmol/g wet wt. cells/min)
______________________________________________________
Sham-operated
Septic
Change (%)
_______________________________________________________________________
Acetoacetate
3-Hydroxybutyrate
Total ketone bodies
Removal of [1-14]oleate
0.52±0.027(6)
1.15±0.062(6)
1.66±0.058(6)
0.55±0.055(6)
0.66±0.056(6) **
1.23±0.077(6) **
+ 6
- 57
- 26
0.55±0.044(6)
0.42±0.053(6)
Esterification of [1-14C]oleate 0.20±0.004(6)
[1-14C]oleate - 14CO2
0.010±0.001(6)
0.19±0.012(6)
- 5
0.015±0.002(6)
+ 50
[1-14C]oleate - 14C acid
0.33±0.028(6)
0.17±0.020(6) *
- 23
- 48
soluble products
_______________________________________________________________________
The rate of formation of acid-soluble (A-S) products, mainly ketone bodies, was
significantly lower in hepatocytes from septic rats (Table 5.1.; Fig. 5.6.) as was the
percentage of [1-14C]oleate converted to these products ( 40±4.5% versus 60±4.8%
; P < 0.05, n=6).
Fig. 5.6.
Metabolism of [1 - C]oleate14
% [1-14C]oleate distribution
100
80
Esterification
A-S products
Metabolic CO 2
60
40
20
0
Sham-op
Septic
Discussion
The concentration of plasma non-esterified fatty acids has been reported
to be increased (Blackburn, 1977) or decreased (Neufeld et al , 1982) by sepsis. In the
present study the slight decrease (13% ; Fig. 3.7. - Chapter 3) was not significant.
Nevertheless, ketogenesis from the added long-chain fatty acid, oleate, in vitro was
markedly depressed (Fig. 5.5.) indicating that the septic liver has a decresed capacity
to produce ketone bodies. Others have shown that perfused livers from rats with
pneumococcal infection produce significantly less ketone bodies from oleate as
compared to perfused livers from fasted controls (Wannemacher et al , 1979).
When octanoic acid was the substrate, isolated perfused livers from
infected rats produced as much ketone bodies as livers from starved controls
(Wannemacher et al , 1979). The same results were found, when butyrate was
used as substrate, in isolated liver cells from caecal-ligated and punctured rats as
compared to hepatocytes from sham-operated rats (Fig. 5.4.)
The fact that ketone body formation remained unchanged when the shortchain fatty acid, butyrate, was the substrate, whereas it was significantly lower in
hepatocytes from septic rats, when the long-chain fatty acid oleate was the substrate,
suggests that a site of inhibition of ketogenesis in the septic liver may be the entry of
long-chain fatty acid into the mitochondria via the carnitine acyl-CoA transferase
system (McGarry & Foster , 1980). Long-chain fatty acyl-CoA would, alternatively, go
into
the
esterification pathway
leading to
formation of triacylglycerols and
phospholipids.
One physiologically important regulatory mechanism of the entry of longchain acylCoA into the mitochondria
is
the modulation of the flux through carnitine palmitoyltransferase I by malonyl-CoA, an
intermediate of the lipogenic pathway. Although the precise mechanism of malonylCoA inhibition on carnitine acyltransferase I in the rat liver mitochondria has not yet
been fully established, available evidence indicates a competitive type of inhibition
against long-chain acyl-CoA substrates (McGarry & Foster, 1980). During starvation, at
the low rates of lipogenesis, decreased malonyl-CoA concentrations permit the
diversion of long-chain fatty acid into oxidative pathways in the mitochondria.
However, despite being starved, hepatocytes from septic rats divert more
long-chain fatty acid to esterification (Fig. 5.6.) than to partial oxidation in the
mitochondria to yield ketone bodies (Fig 5.5.).
The rate of hepatic lipogenesis in vivo is significantly increased (Fig. 6.2. Chapter 6) in septic rats. Although lipogenesis is considered to be positively correlated
with cytosolic malonyl-CoA concentrations it must be emphasized that hepatic carnitine
acyl-CoA transferase I (CAT I) is less sensitive to inhibition by malonyl-CoA in livers
from starved rats (Cook et al , 1980). In addition, lipogenesis has not been measured in
the present hepatocyte experiments.
Sepsis induced by injection of bacteria has been reported not to increase
hepatic malonyl-CoA (Wannemacher et al , 1979) or alternatively to increase this
metabolite (Vary et al , 1986). There is also evidence that the sensitivity of carnitine
acyl transferase I to malonyl-CoA can be rapidly increased by insulin (Gamble & Cook,
1985) and, therefore, it may be pertinent that our septic rats had higher plasma insulin
concentration ( Fig. 3.5. - Chapter 3).
Decreased [D-3-hydroxybutyrate]/[acetoacetate] ratio (Table 5.1.) together
with the lower concentrations of acetyl-CoA ( Fig. 3.17. - Chapter 3) in livers from
septic rats support the view of depressed ∫-oxidation in sepsis, as a result of the
diminished flux of long-acylCoA into the mitochondria.
The reason for the impairment of ketogenesis in livers from septic rats,
therefore, appears to be multifactorial and involves a possible decrease in hepatic
uptake of non-esterified fatty acids, depression of the entry of long-chain acyl-CoA into
the mitochondria for ∫-oxidation and lower hepatic concentration of acetyl-CoA. The
increase in the percentage of metabolic CO2 formation, by hepatocytes
from septic rats, may be another contributory factor for it
indicates more acetyl-CoA
entering the Krebs cycle for oxidation, thus decreasing the availability of acetyl-CoA as
the immediate cause for the decreased ketogenesis.
CHAPTER 6
Time course of changes in rates of lipogenesis and hepatic cholesterogenesis in vivo
after the induction of sepsis
Introduction
Despite the fact lipids constitute one of the principal sources of body energy and
sepsis produces various alterations in fat metabolism, many of the metabolic changes
induced by sepsis still need clarification. More than six decades have passed since the
changes in concentrations of cholesterol in serum, during bacterial infection, were first
described (Kipp, 1920). However, little is known of the regulation of cholesterol
metabolism during sepsis.
Some information has recently been collected on the metabolism of non-esterified
fatty acids during infection (Blackburn, 1977; Wannemacher et al , 1979; Beisel &
Wannemacher, 1980). The availability of non-esterified fatty acids may be decreased in
sepsis as a result of the anti-lipolytic effect of the hyperinsulinaemia (Fig. 3.5. - Chapter 3),
to suppress hormone sensitive lipase activity (Steinberg & Khoo, 1977) resulting in lower
concentrations of non-esterified fatty acids, as reported in various models of sepsis (Beisel
& Wannemacher, 1980). The plasma concentration of non-esterified fatty acids, however,
was not significantly changed in the present studies (Fig. 3.7. - Chapter 3).
A decrease in the percentage of non-esterified fatty acid uptake (23%) was found
in isolated hepatocytes from caecal-ligated and punctured rats (Table 5.1. - Chapter 5).
Within hepatocytes, non-esterified fatty acids appear to be preferentially esterified in the
livers of septic rats, rather than partially or totally oxidized with formation of ketone
bodies or metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al , 1979). The
percentage of [1-14C]oleate esterified was significantly higher in isolated hepatocytes from
caecal- ligated rat as compared to that of hepatocytes from sham-operated rats (Fig. 5.6. Chapter 5). As a result of decreased oxidation of long-chain fatty acids the rate of
ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the hyperketonaemia, associated with
starvation, decreased in sepsis (Fig. 3.8. - Chapter 3). Non-esterified fatty acid utilization
by the liver, therefore, appears to be decreased in experimental or clinical sepsis with the
development of an elevation in plasma concentrations of triacylglycerol (Levin et al ,
1972; Kaufmann et al , 1976; Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al
, 1981 Alvares & Ramos, 1986), and fatty infiltration of the liver (Mukherjee et al , 1973;
Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of increased
triacylglycerol synthesis accompanied by a decreased or insufficient production of the
proteins needed to export very low density lipoprotein (VLDL) as suggested by Forse &
Kinney (1985). However, general protein synthesis in the liver has been reported to be
stimulated by sepsis (Long et al , 1977; Hasselgen et al, 1984). Decreased plasma
clearance of triacylglycerol as a result of diminished activity of lipoprotein lipase has been
reported as contributory to the hypertriacylglycerolaemia induced by sepsis (Kaufmann et
al , 1976; Robin et al , 1981; Lanza-Jacoby et al , 1982).
Hepatic lipogenesis in vitro , measured by the incorporation of [1-14C]acetate
into lipids in hepatocytes isolated from rats with
pneumococcal infection, is increased in response to sepsis (Canonico
et al , 1977).
There appears to be no information, however, on rates of lipid synthesis in vivo in the liver
or adipose tissue after induction of sepsis.
Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated rats
(Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats (Paceet al
,1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from rats with
pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the flow of acetylCoA toward lipogenesis and cholesterolegenesis in vivo rather than to ketogenesis or
oxidation in the Krebs tricarboxylic acid cycle (Fig. 6.1.).
Fig. 6.1.
Total cholesterol concentration in serum has been reported to increase, decrease,
or remain unchanged during sepsis (Fiser et al , 1971 and 1972; Royle & Kettlewell, 1980;
Alvares & Ramos, 1986). Such variation in cholesterol concentration provides little
information on the dynamics involved in cholesterol synthesis, transport and utilization.
Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic acid, showed
increased incorporation of the labelled mevalonate into free cholesterol in plasma when
compared to control monkeys (Fiser et al , 1971). In vitro , [14C]acetate incorporation into
cholesterol by isolated hepatocytes from rats with pneumococcal infection was
significantly increased as compared to the incorporation by hepatocytes from control rats
(Cannonico et al , 1977). However, there appears to be no information on the rates of
hepatic cholesterol synthesis in vivo in response to sepsis.
The entero-hepatic circulation of bile acids plays an essential role in the absorption
of cholesterol from the intestine and in the regulation of cholesterol metabolism in the liver
(for review see : Myant, 1981 and Gibbons et al , 1982). The diversion of bile acids, or the
complete interruption of the bile duct, leads to the absence of bile acids in the intestine,
which in turn, supresses the normal uptake of cholesterol from the intestinal lumen. There
is some evidence that it is the transport of intestinal cholesterol to the liver which directly
determines the rates of hepatic cholesterol synthesis (Weis & Dietschy, 1975). This view is
supported by the fact that restoration of the entero-hepatic circulation, in rats subjected to
biliary diversion, failed to prevent the rise in hepatic cholesterogenesis, whereas, the
infusion of chylomicron cholesterol led to a prevention in the rise of hepatic sterol
synthesis rates (Gibbons et al , 1982). In addition, bile acids inhibit cholesterol 7Ïhydroxylase (the rate-limiting enzyme in the process of bile acids formation from
cholesterol) and, therefore, would
prevent the catabolism and removal of cholesterol from the liver, resulting in suppression
of HMG-CoA reductase activity (Nervi & Dietschy, 1978). The possibility of bile acids
having a direct effect on HMG-CoA reductase, in the liver, would suggest diversion rather
than obstruction of the bile duct as the resonable approach to study the metabolism of
cholesterol in the liver. However, opposing the idea of a direct bile acid effect is the fact
that partial or complete biliary obstruction causes an increase, rather than the expected
decrease, in hepatic cholesterol synthesis (Fredrickson et al , 1954; Gibbons et al , 1982).
To investigate the time course of changes in vivo in lipogenesis in the liver, and in
white and brown adipose tissue, and hepatic cholesterogenesis, after the induction of
sepsis, caecal-ligated and punctured rats were studied. Sham-operated rats were used as
controls. As the absorption of bile acids and cholesterol in the intestinal lumen of caecalligated and punctured rats may be impaired, due to the development of an adynamic ileus
in these rats, ligation of the bile duct of a group of septic and sham-operated rats was also
performed. This complete interruption of the entero-hepatic circulation of bile acids may
help to evaluate the contribution of the impairment in the absorption of cholesterol and bile
acids from the gut to hepatic cholesterogenesis in sepsis.
Experimental Design
Rats, after being subjected to caecal-ligation and puncture or sham-operation, were
fasted for 12, 24 or 48 h before livers were removed and studied (Fig.6.2.).
Fig. 6.2.
For details of the operations and the techniques involved in the measurement of
lipogenesis, cholesterogenesis, preparation of microsomes and the measurement of
hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and lipoprotein lipase
activities see Chapter 2. A group of rats had their bile duct ligated immediately before
either caecal-ligation and puncture or sham-operation was performed (Chapter 2).
Results
The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo was
significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48 h
after the operations, as compared to that of livers from sham-operated animals (Fig. 6.3.).
Fig. 6.3.
Rate of H
3
incorporation
O
into fatty acids
2
**
(7)
(4)
µmol/g wet wt. liver/h
4.0
3.0
*
(18)
Sham-op
Septic
(3)
(13)
2.0
* P < 0.05
** P < 0.001
(6)
1.0
X + S.E.M.
0.0
12
24
48
Increased rates of formation of total non-saponifiable lipids in vivo were also
found to be present in livers from septic rats when compared to those found in livers from
sham-operated rats (Fig. 6.4.) as early as at 12 h after the operation.
Fig. 6.4.
Rate of H3 incorporation
into non-saponifiable lipids
2O
*
(4)
7
6
Sham-op
Septic
**
(7)
5
**
(18)
4
3
* P < 0.05
(3)
(6)
** P < 0.001
2
(13)
X + S.E.M.
1
0
12
24
48
Therefore, livers from septic rats have significantly increased rates of total
lipogenesis as compared to those of sham-operated rats. This increase in lipid synthesis
appears to occur both in rates of formation of saponifiable (fatty acids) and nonsaponifiable (squalene, cholesterol, other sterols) fractions (Fig. 6.5.).
Fig. 6.5.
Rate of H
10
3
incorporation
into lipids
2O
*
*
8
*
6
saponified
non-saponified
4
2
* P < 0.001
0
sham
septic
1
2
12 h
sham
septic
3
4
24 h
sham
septic
5
6
48 h
Effects of sepsis on squalene and cholesterol synthesis in vivo and hydroxymethylglutarylCoA reductase activity in the liver
The non-saponifiable fraction was separated into the various sterol fractions.
Sepsis produced a significant increase in the rate of squalene formation in the liver at 24 h
after the operation (Fig. 6.6.). No change in rate was observed at the other time points
studied.
Fig. 6.6.
Rate of H
3
incorporation
into squalene
O
2
*
(7)
(4)
0.50
(18)
0.40
(3)
Sham-op
Septic
(13)
0.30
(6)
0.20
* P < 0.05
0.10
X + S.E.M.
0.00
12
24
48
Starvation caused the incorporation of 3H2O into cholesterol to decrease with time
in both sham-operated and caecal-ligated and punctured rats. However, the rates of
cholesterol synthesis in the livers of septic rats in vivo were significantly higher than those
found in the livers of sham-operated rats. This increase (2.3 fold) occurred as early as 12 h
after the operations reaching a four-fold increase at 48 h (Fig. 6.7.).
Fig. 6.7.
Rate of H
3incorporation
2O
into cholesterol
*
(4)
4.0
sham-op
septic
3.0
2.0
**
(11)
**
(18)
(3)
* P < 0.05
** P < 0.001
(11)
1.0
(12)
X + S.E.M.
0.0
12
24
48
The total activity of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the
livers of septic rats was significantly increased compared to that found in livers of shamoperated rats at 48 h after the operations (Fig. 6.8.).
Fig. 6.8.
Total HMG-CoA reductase activity
pmol mevalonate/mg protein/min
*
(7)
20
sham-op
septic
10
(4)
* P < 0.02
X + S.E.M.
0
48 h
HMG-CoA reductase is phosphorylated by a protein kinase and phosphorylation
leads to inactivation of the enzyme (Gibbons et al , 1982; Gibson & Parker, 1987). The
activity of both the phosphorylated ('inactive' form) and the non-phosphorylated or
expressed form of this enzyme (Fig. 6.9.) was measured in livers from septic and shamoperated rats at 48 h after the operation. Sepsis induced no appreciable change in the
expressed activity of HMG-CoA reductase (non-phosphorylated or 'active'). As there was a
significant increase in the total enzyme activity (Fig. 6.8.) this was mainly due to an
increase in activity of the phosphorylated or 'inactive' form of the enzyme (Fig. 6.9.).
Fig. 6.9.
*
(7)
25
20
Sham-op
Septic
15
10
* P < 0.01
(7)
(4)
(4)
5
X + S.E.M.
( ) Nº of observations
0
'active'
1
phosphorylated
2
It may be of note to mention that the technique of rapid sampling of the liver for the
preparation of liver microsomal fraction, as described by Easom & Zammit (1984; 1986),
was not used in the present studies, and, therefore, the measured activity of the
dephosphorylated or 'active' form of the enzyme may not correspond to the actual total
enzyme activity. Nevertheless, the same sampling technique was used for both septic and
sham-operated rats.
Effects of sepsis on lipoprotein lipase activity and rates of lipogenesis in vivo in adipose
tissue
In order to obtain some information on the changes in lipid metabolism in adipose
tissue, and to compare to those found in the liver, rates of lipogenesis and lipoprotein
lipase activity were measured in white and brown adipose tissue of septic and shamoperated rats. Sepis caused no appreciable change in the rate of total lipid synthesis in
white adipose tissue at 12 h after the operation, however, in contrast to the findings in the
liver, sepsis decreased the lipogenic rate at 48 h in adipose tissue (Fig. 6.10.) when
compared to that of sham-operated rats.
Fig. 6.10.
The rate of lipogenesis in brown adipose tissue of septic rats was significantly
decreased when compared to that of sham-operated rats, at 12 h after the operations.
However, at 48 h, brown adipose tissue from septic rats synthesized lipids at similar rates
to those of sham-operated rats (Fig.6.11.).
Fig. 6.11.
Lipoprotein lipase activity was measured in adipose tissue from septic and shamoperated rats at 48 h after the operations. Sepsis did not cause appreciable change in
lipoprotein lipase activity in white adipose tissue (Septic : 0.302±0.035 , n=9 ; Shamoperated : 0.253±0.048 , n=8 ; mean values ± S.E.M.; enzyme activity expressed as nmol
of FFA released/min/mg of tissue). However, this enzyme was significantly more active in
brown adipose tissue from septic rats at 48 h ( Septic : 1.37±0.26 , n=9 ; Sham-operated
: 0.61±0.16 , n=8 ; P < 0.05 - Student t-test).
The effect of the complete interruption of the entero-hepatic circulation of bile acids
accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver in
vivo
The complete interruption of the entero-hepatic circulation of bile acids obtained
by bile duct ligation (BDL) produced a significant increase in the rates of
cholesterolegenesis in livers of sham-operated+BDL rats as compared to those of shamoperated rats at 48 h after the operations. There was, however, no significant further
increase in the rates of cholesterol synthesis in the livers of septic rats+BDL as compared
to caecal-ligated and punctured rats (Fig. 6.12.).
Fig. 6.12.
There was no appreciable effect of bile duct ligation (BDL) on the rate of fatty
acid synthesis in sham-operated animals (Sham-op+BDL : 1.95±0.13, n=4 ; Sham-op :
1.95±0.24, n=13; 3H2O
incorporation expressed as µmol of labelled fatty acid/g wet wt. liver/h). The stimulatory
effect produced by bile-duct ligation was, therefore, specific for cholesterol synthesis.
It is important to add that caecal-ligated and punctured rats which also had bile
duct ligation were hyperinsulinaemic, as compared to sham-operated rats which had their
bile duct ligated (Septic+BDL : 35.5±6.02 (n-4); Sham-operated : 18.7±1.76 (n=4) - mean
values ± S.E.M. expressed as µunit/ml of plasma ; P < 0.05 - Student t test).
Discussion
Hypertriacylglycerolaemia is a common finding during sepsis (Fig. 3.6. - Chapter
3) and has been thought to be a consequence of both increased triacylglycerol production
and decreased triacylglycerol clearance from the circulation (Kaufman et al , 1976; Robin
et al , 1981). Hepatic esterification of [1-14C]oleate was increased by this model of sepsis
(Fig. 5.6. - Chapter 5) and this was accompanied by unchanged lipoprotein lipase activity
in white adipose tissue from septic rats at 48 h after the operation. In contrast, lipoprotein
lipase activity has been reported to be decreased by infection (Robin et al , 1981; LanzaJacoby et al , 1982). A dose-dependent suppression of lipoprotein lipase activity when
adipocytes were exposed either to purified recombinant interleukin-1 (Price et al , 1986) or
to cachectin (Price et al , 1986) has been shown in vitro .
The in vivo rates of lipogenesis in white adipose tissue, however, were decreased
by sepsis (Fig. 6.10.) at 48 h after the operation. Pekala et al , have shown that crude
preparations of cachectin were able to suppress the activity of key lipogenic enzymes such
as acetyl-CoA carboxylase and fatty acid synthetase in the white adipocytes, which in turn
would probably inhibit lipogenesis. This supports the view that "hormones" in the crude
medium from endotoxin-stimulated macrophages are capable of promoting important
changes in the metabolism of the adipose cell (Price et al , 1986).
Sepsis did not induce appreciable change in the rate of lipid synthesis in brown adipose
tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase activity was, however,
significantly increased in this tissue in septic rats, at the same time point, suggesting the
available triacylglycerol is being oxidised and used for chemical energy or heat production.
Although high rates of lipogenesis may be necessary for the production of sufficient
amounts of non-esterified fatty acids for the activation of thermogenin (Cannon &
Nedergaard, 1985), thermogenesis can also occur without stimulated lipogenesis, the two
being regulated independently (Himms-Hagen, 1985). Sepsis caused a significant increase
in the rate of hepatic production of lipids in vivo at all time points studied (Fig. 6.5.).
There was increased production not only of fatty acids, represented by the saponifiable
lipid fraction (Fig. 6.3.) but also in the total non-saponifiable lipid fraction (Fig. 6.4.).
During sepsis many factors create an increased demand for cholesterol, including
increased hepatic production of lipoproteins (necessary to complement the increased
triacylglycerol synthesis) and possibly increased cell membrane synthesis in tissues
involved with defence and repair. As this need for cholesterol cannot be met from dietary
sources because the gut is not always available to absorb nutrients for example during
intra-abdominal sepsis, endogenous synthesis within the liver is expected to increase. In
fact, cholesterol synthesis was significantly increased in vivo in the livers of septic rats as
early as 12 h after the operation (Fig. 6.7.), whereas squalene synthesis was increased only
at 24 h (Fig. 6.6.).
Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.5. & 6.7.)
accompanied by decreased rates of ketogenesis (see Chapter 5), in the presence of
diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest sepsis
directs the flow of acetyl-CoA preferentially toward lipid and cholesterol synthesis rather
than to partial oxidation, with ketone body production, or complete oxidation in the
tricarboxylic acid cycle.
As absorption of bile acids and cholesterol may be partially impaired in this model
of sepsis, due to the presence of an adynamic ileus, in response to the localized peritonitis,
this may be a contributory factor to the raised cholesterol synthesis in the livers of septic
rats. Although hepatic fatty acid synthesis was not affected by bile duct ligation (BDL) in
sham-operated rats, at 48 h after the operation, cholesterol synthesis rates in the livers of
these rats were increased to levels comparable to those of septic rats (Fig. 6.12.). However,
rats subjected to subcutaneous bacterial injection (Canonico et al , 1977), and therefore
less likely to develop an adynamic ileus, had increased rates of cholesterogenesis in vitro .
This finding does not support the view that partial interruption of the bile acid hepatoenteric circulation is the main reason for the increase in cholesterol synthesis in vivo ,
found in this model of sepsis, and therefore favours an independent effect of sepsis.
Septic rats had increased total HMG-CoA reductase activity (Fig. 6.9.) associated
with augmented cholesterol synthesis (Fig. 6.8.). There was, however, no change in the
activity of the 'active' or expressed form of the enzyme (Fig. 6.10.). This is perhaps
surprising in view of the fact that insulin, at least in the fed state, is considered to rapidly
increase the proportion of this enzyme in the active form (Easom & Zammit, 1986).
Insulin, present in higher concentrations in the plasma of septic rats at all time points
studied (Fig. 3.5. - Chapter 3), may have exerted its longer-term effect by increasing the
synthesis of this enzyme rather than changing the activation state.
The increased rates of hepatic lipogenesis, induced by caecal-ligation and
puncture, give support to the view of an inhibition in the entry of long-chain acyl-CoA in
the mitochondria of hepatocytes from septic rats by higher concentrations of malonyl-CoA,
an intermediate in the lipogenic pathway (see Chapter 5). This stimulation in the rates of
hepatic lipogenesis and cholesterogenesis by sepsis may well be due to greater availability
of lactate (Figs. 3.2. & 3.9. - Chapter 3) and pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the
blood and liver, and the presence of hyperinsulinaemia (Fig. 3.5. - Chapter 3).
CHAPTER 6
Time
course
of
changes
in
lipogenesis
and
hepatic
cholesterolegenesis in vivo after the induction of sepsis
Introduction
Despite the fact lipids constitute one of the principal sources of body energy
and sepsis produce various alterations on fat metabolism, many of these metabolic
changes induced by sepsis still need clarification. More than six decades have
passed since the changes in concentrations of cholesterol in serum, during bacterial
infection, were first described (Kipp, 1920). However, little is known on the regulation
of cholesterol metabolism during sepsis.
Some information has recently been collected on the metabolism of nonesterified fatty acids during infection.The availability of non-esterified fatty acids may
be decreased in sepsis as a result of the inhibitory effect of hyperinsulinaemia (Fig.
3.5. - Chapter 3) on lypolysis , suppressing hormone sensitivity lipase activity
(Steinberg & Khoo, 1977) resulting in lower concentrations of non- esterified fatty
acids, as reported in various models of sepsis (Beisel & Wannemacher, 1980).
Plasma concentration of non esterified fatty acids, however, remained unchanged in
the present studies (Fig. 3.7. - Chapter 3).
A decrease in the percentage of non-esterified fatty acid uptake (23%) was
found in livers from caecal ligated and punctured rats (Table 5.1. - Chapter 5). Once
in the
hepatocytes, non-esterified fatty acids suffer preferential esterification in the livers of
septic rats, rather than partial or total oxidation with formation of ketone bodies or
metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al , 1979). The
percentage of [1-14C]oleate esterified was significantly higher in isolated
hepatocytes from caecal- ligated rat as compared to that of hepatocytes from shamoperated rats (Fig. 5.6. - Chapter 5). As a result of decreased oxidation of long-chain
fatty acids the rate of ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the
hyperketonaemia, in adaptation to starvation, abolished by sepsis (Fig. 3.8. - Chapter
3). Non-esterified fatty acid utilization by the liver, therefore, appears to be decreased
in experimental or clinical sepsis with the development of elevation in plasma
concentrations of triacylglycerols (Levin et al , 1972; Kaufmann et al , 1976;
Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al , 1981 Alvares & Ramos,
1986), and fatty infiltration of the liver (Mukherjee et al , 1973; Pace et al , 1977;
Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of
increased triacylglycerol synthesis accompanied by a decreased or insuficient
production of the proteins needed to export very low density lipoprotein (VLDL) as
suggested by Forse & Kinney (1985). However, protein synthesis in the liver has
been reported to be stimulated by sepsis (Long et al , 1979; Hasselgen et al, 1984).
Decreased plasma clearence of triacylglycerol as a result of diminished activity of
lipoprotein lipase has been reported as contributory to the hypertriacylglycerolaemia
induced by sepsis (Kaufmann et al , 1976; Robin et al , 1981; Lanza-Jacoby et al ,
1982).
Hepatic lipogenesis in vitro , measured by the incorporation of [114C]acetate into lipids in hepatocytes isolated from rats with pneumococcal infection,
is increased in response to sepsis (Canonico et al , 1977). There appears
to be no information, however, on rates of lipid synthesis in vivo in the liver or
adipose tissue after induction of sepsis.
Total cholesterol concentration in serum has been reported
to increase,
decrease, or remain unchanged during sepsis (Lindell et al , 1964; Fiser et al , 1971;
information into the dynamics involved in cholesterol synthesis, transport and
utilization. Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic
acid, showed increased incorporation of the labelled mevalonate into free cholesterol
in plasma when compared to control monkeys (Fiser et al , 1971). In vitro ,
[14C]acetate incorporation into cholesterol by isolated hepatocytes from rats with
pneumococcal infection was significantly increased as compared to the incorporation
by hepatocytes from control rats (Cannonico et al , 1977). However, there appears
to be no information on the rates of hepatic cholesterol synthesis in vivo in response
to sepsis.
Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated
rats (Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats
(Pace et al , 1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from
rats with pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the
flow of acetyl-CoA toward lipogenesis and cholesterolegenesis in vivo rather than to
ketogenesis or oxidation in the Krebs tricarboxilic acid cycle.
To investigate the time course of changes in vivo in lipogenesis in the liver
and white and brown adipose tissue, and hepatic cholesterolegenesis, after the
induction of sepsis, caecal-ligated and punctured rats were studied. Sham-operated
rats were used as controls.
Experimental Design
Rats, after being subjected to caecal-ligation and puncture or shamoperation, were fasted for 12, 24 or 48 h before livers were removed and studied
(Fig.6.1.).
Fig. 6.1.
OP
12 h
24 h
48 h
Rats
3 H O injected
2
Livers removed
Lipid exctraction
Lipogenesis
Cholesterolegenesis
Microsomal preparation
HMGCoA reductase activity
For details on the operations and the techniques involved in the
measurement of lipogenesis, cholesterolegenesis, preparation of microsomes and
the measurement of hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and
lipoprotein lipase activities see Chapter 2. A group of rats had their bile duct ligated
immediately before either caecal-ligation and puncture or sham-operation was
performed (Chapter 2).
Results
The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo
was
significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48
h after the
operations, as compared to that of livers from sham-operated animals (Fig. 6.2.).
Fig. 6.2.
Rate of fatty acid synthesis in the liver
**
(7)
(4)
µmol fatty acid/g wet wt. liver/h
4.0
3.0
*
(18)
Sham-op
Septic
(3)
(13)
2.0
(6)
* P < 0.05
1.0
** P < 0.001
( nº of observations )
0.0
12
24
48
Time after the operations (h)
found to be present in livers from septic rats when compared to those found in livers
from sham operated rats (Fig. 6.3.) as early as at 12 h after the operations.
Fig. 6.3.
Rate of non-saponifiable lipid synthesis
*
(4)
7
µmol lipid/gwet wt. liver/h
6
**
(7)
5
**
(18)
4
3
(3)
(6)
Sham-op
Septic
* P < 0.05
2
** P < 0.001
(13)
1
0
12
24
48
Therefore, livers from septic rats exhibit rates of total lipogenesis significantly
increased as compared to those of sham-operated rats. This increase in lipid
synthesis appears to be at the expense of increases in rates of formation of both
saponifiable (fatty acids) and non-saponifiable (squalene, cholesterol, other sterols)
fractions (Fig. 6.4.).
Fig. 6.4.
Rate of total lipogenesis in the liver
µmol lipid formed/g wet wt. liver/h
10
*
*
8
*
6
saponified
non-saponified
4
2
* P < 0.001
0
sham
1
septic
2
12 h
sham
septic
3
4
24 h
sham
septic
5
6
48 h
Effects of sepsis on lipoprotein lipase activity and rates of total lipogenesis in vivo in
adipose tissue
Depite inducing no appreciable change in the rate of total lipid synthesis in
white adipose tissue at 12 h after the operation, sepsis did decrease the total
lipogenic rate at 48 h (Fig. 6.5.) when compared to that of sham-operated rats.
Fig. 6.5.
8
(8)
7
µmol/g wet wt. tissue/h
6
5
(10)
(7)
*
(11)
4
sham-op
septic
3
2
* P < 0.05
1
0
12
48
The rate of total lipogenesis in brown adipose tissue of septic rats was
significantly decreased when compared to that of sham-operated rats, at 12 h after
the operations. However, at 48 h, brown adipose tissue from septic rats produced
lipids at similar rates to those of sham-operated rats (Fig.6.6.).
Fig. 6.6.
µmol/g wet wt. tissue/h
20
(7)
*
(8)
10
(10)
(12)
sham-op
septic
* P < 0.05
0
12
48
Lipoprotein lipase activity was measured in adipose tissue from septic and
sham-operated rats at 48 h after the operations. Sepsis did not cause appreciable
change in lipoprotein lipase activity in white adipose tissue (septic : 0.302±0.035 ,
n=9 ; sham-op : 0.253±0.048 , n=8 ; mean±SEM with number of observations in
parenthesis). However, this enzyme was significantly more active in brown adipose
tissue from septic rats at 48 h (septic : 1.37±0.26 , n=9 ; sham-op : 0.61±0.16 , n=8 ;
P < 0.05 - Student t-test).
Effects of sepsis on squalene and cholesterol synthesis in vivo
and
hydroxymethylglutaryl-CoA reductase activity in the liver
Sepsis produced significant increase in the rate of squalene formation in the
liver at 24 h after the operations (Fig. 6.7.). No effect was observed at the other time
points studied.
Fig. 6.7.
Rate of squalene synthesis in the liver
µmol squalene/g wet wt. liver/h
*
(7)
(4)
0.50
(18)
0.40
(3)
Sham-op
Septic
(13)
0.30
(6)
0.20
* P < 0.05
0.10
0.00
12
24
48
The rates of cholesterol synthesis in the livers of septic rats in vivo were
significantly higher than those found in the livers of sham-operated rats. This
increase (2.3 fold) occured as early as 12 h after the operations reaching a four-fold
increase at 48 h (Fig. 6.8.).
Fig. 6.8.
Rate of cholesterol synthesis
*
(4)
µmol formed/g wet wt. liver/h
4.0
3.0
2.0
**
(11)
**
(18)
(3)
* P < 0.05
(11)
1.0
sham-op
septic
(12)
** P < 0.001
( nº of operations )
0.0
12
24
48
livers of septic rats was significantly increased compared to that found in livers of
sham-operated rats at 48 h after the operations (Fig. 6.9.).
Fig. 6.9.
*
(7)
20
sham-op
septic
10
(4)
* P < 0.02
0
48 h
HMG-CoA reductase is phosphorylated by a protein kinase. Phosphorylation
leads to inactivation of the enzyme. We have measured the activity of both the
phosphrylated ('inactive' form) and the non-phosphorylated or expressed form of this
enzyme (Fig. 6.10.) in livers from septic and sham-operated rats at 48 h after the
operations. Sepsis induced no appreciable change in the expressed activity of HMGCoA reductase (non-phosphorylated or 'active'). As there was a significant increase
in the total enzyme activity (Fig. 6.9.) this was mainly due to an increase in activity of
the phosphorylated or 'inactive' form of the enzyme (Fig. 6.10.).
Fig. 6.10.
18
*
(7)
16
14
12
10
6
sham-op
septic
(7)
8
(4)
4
(4)
2
* P < 0.01
0
'active'
phosphorilated
48 h after the operations
accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver
in vivo
The complete interruption of the entero-hepatic circulation of bile acids
obtained by bile duct ligation (BDL)
produced a significant increase in the rates of cholesterolegenesis in livers of shamoperated+BDL rats as compared to those of sham-operated rats at 48 h after the
operations. There was, however, no significant further increase in the rates of
cholesterol synthesis in the livers of septic rats+BDL as compared to caecal-ligated
and punctured rats (Fig. 6.11.).
Fig. 6.11.
Hepatic cholesterol synthesis and BDL
4
†
µmol formed/g wet wt. liver/min
(4)
3
(8)
(18)
2
1
sham-op
septic
(12)
† P < 0.001
° sham-op/no BDL
0
No 1BDL
BDL
2
48 h after the operations
There was no appreciable effect of BDL on the rate of fatty acid synthesis on shamoperated animals (sham-op+BDL : 1.95±0.13, n=4 ; sham-op : 1.95±0.24, n=13).
This stimulatory effect produced by bile-duct ligation was, therefore, specific for
cholesterol synthesis as found by Fredrickson et al , 1953.
Discussion
Hypertryacylglycerolaemia is a comon finding during sepsis (Fig. 3.6. Chapter 3) and has been thought to be a consequence of both increased
tryacylglycerol production
and decreased tryacylglycerol clearence from the
circulation (Robin et al , 1981). Hepatic esterification of [1-14C]oleate was increased
by this model of sepsis (Fig. 5.6. - Chapter 5) and this was accompanied by
unchanged lipoprotein lipase activity in white adipose tissue from septic rats at 48 h
after the operations (Fig. 6.5.). The rate of lipogenesis in white adipose tissue,
however, was decreased by sepsis (Fig. 6.5.) at the same time point studied.
Sepsis did not induce appreciable change in the rate of lipid synthesis in
brown adipose tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase
activity was, however, significantly increased in this tissue in septic rats, at the same
time point studied, suggesting the hydrolysed tryacylglycerol is being oxidised and
used for energy or heat production rather than for lipogenesis. Although high rates of
lipogenesis may be necessary for the production of sufficient amount of nonesterified fatty acids for the activation of thermogenin (Cannon & Nedergaard, 1985),
thermogenesis can also occur without stimulated lipogenesis, the two being
regulated independently (Himms-Hagen, 1985).
Sepsis caused significant increase in the rate of hepatic production of lipids
in vivo at all time points studied (Fig. 6.4.). There was increased production not only
of fatty acids, represented by the saponifiable lipid fraction (Fig. 6.2.) but also in the
total non-saponifiable lipid fraction (Fig. 6.3.).
During sepsis many factors create a large demand for cholesterol, including
increased hepatic production of lipoproteins (necessary to match with the increased
triacylglycerol synthesis) and possibly increased cell membrane synthesis at tissues
involved with defence and repair. As this need for cholesterol can not be met from
dietary sources because the gut is not always available to absorb nutrients e.g.
during intra-abdominal sepsis, endogenous synthesis within the liver is expected to
increase. In fact, cholesterol synthesis was significantly increased in vivo in the
livers of septic rats as early as at 12 h after the operations (Fig. 6.8.) while squalene
synthesis was increased at 24 h (Fig. 6.7.).
Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.4 & 6.8.)
diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest
sepsis directs the flow of acetyl-CoA
preferentially toward lipid and cholesterol
synthesis rather than to partial oxidation with ketone body production or complete
oxidation in the tricarboxylic acid cycle.
As absorption of bile acids may be partially impaired in this model of sepsis
due to the presence of an adynamic ileus, in response to the localized peritonitis, this
may be a contributory factor to the raised cholesterol synthesis in the livers of septic
rats. Although hepatic fatty acid synthesis was not affected by bile duct ligation (BDL)
in sham-operated rats at 48 h after the operation, cholesterol synthesis in the livers
of these rats was increased to level comparable to those of septic rats (Fig. 6.11.).
However, rats subjected to subcutaneous bacterial injection (Canonico et al , 1977),
and therefore less likely to develop an adynamic ileus, presented increased rates of
cholesterolegenesis in vitro , questioning the idea of the partial interruption of the bile
acid hepato-enteric circulation as the main reason for the increase in cholesterol
synthesis in vivo , found in this model, and favouring an independent effect of sepsis.
Hydroxymethylgluratyl-CoA reductase activity and cholesterolegenesis were
measured in the same animals. Septic rats presented with higher total enzyme
activity (Fig. 6.9.) along with aumented cholesterol synthesis (Fig. 6.8.). There was ,
however , no change in the activity of the 'active' or expressed form of the enzyme
(Fig. 6.10.). Insulin, present in higher concentrations in the plasma of septic rats at all
time points studied (Fig. 3.5. - Chapter 3) may have exerted its longer-term effect by
increasing this enzyme synthesis rather than changing the activation state of the
enzyme.
The increased rates of hepatic lipogenesis induced by caecal-ligation and
puncture give support to the view of an inhibition in the entry of long-chain acyl-CoA
in the mitochondria of hepatocytes from septic rats by higher concentrations of
malonyl-CoA, an intermediate in the lipogenic pathway (see Chapter 5). This
stimulation in the rates of hepatic lipogenesis and cholesterolegenesis by sepsis may
well be due to greater availability of lactate (Figs. 3.2. & 3.9. - Chapter 3) and
pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the blood and liver, and the presence of
hyperinsulinaemia (Fig. 3.5. - Chapter 3).
CHAPTER 6
Time course of changes in rates of lipogenesis and hepatic
cholesterolegenesis in vivo after the induction of sepsis
Introduction
Despite the fact lipids constitute one of the principal sources of body energy
and sepsis produce various alterations on fat metabolism, many of these metabolic
changes induced by sepsis still need clarification. More than six decades have
passed since the changes in concentrations of cholesterol in serum, during bacterial
infection, were first described (Kipp, 1920). However, little is known on the regulation
of cholesterol metabolism during sepsis.
Some information has recently been collected on the metabolism of nonesterified fatty acids during infection (Blackburn, 1977; Wannemacher et al , 1979;
Beisel & Wannemacher, 1980).The availability of non-esterified fatty acids may be
decreased in sepsis as a result of the anti-lipolytic effect of the hyperinsulinaemia
(Fig. 3.5. - Chapter 3), to suppress hormone sensitive lipase activity (Steinberg &
Khoo, 1977) resulting in lower concentrations of non-esterified fatty acids, as
reported in various models of sepsis (Beisel & Wannemacher, 1980). Plasma
concentration of non-esterified fatty acids, however, was not significantly changed in
the present studies (Fig. 3.7. - Chapter 3).
A decrease in the percentage of non-esterified fatty acid uptake (23%) was
found in isolated hepatocytes from caecal ligated and punctured rats (Table 5.1. Chapter 5).
Whithin hepatocytes, non-esterified fatty acids appear to be preferentially esterified in
the livers of septic rats, rather than partially or totally oxidized with formation of
ketone bodies or metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al ,
1979). The percentage of [1-14C]oleate esterified was significantly higher in isolated
hepatocytes from caecal- ligated rat as compared to that of hepatocytes from sham-
operated rats (Fig. 5.6. - Chapter 5). As a result of decreased oxidation of long-chain
fatty acids the rate of ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the
hyperketonaemia, associated with starvation, decreased by sepsis (Fig. 3.8. Chapter 3). Non-esterified fatty acid utilization by the liver, therefore, appears to be
decreased in experimental or clinical sepsis with the development of elevation in
plasma concentrations of triacylglycerols (Levin et al , 1972; Kaufmann et al , 1976;
Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al , 1981 Alvares & Ramos,
1986), and fatty infiltration of the liver (Mukherjee et al , 1973; Pace et al , 1977;
Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of
increased triacylglycerol synthesis accompanied by a decreased or insuficient
production of the proteins needed to export very low density lipoprotein (VLDL) as
suggested by Forse & Kinney (1985). However, genearal protein synthesis in the
liver has been reported to be stimulated by sepsis (Long et al , 1979; Hasselgen et
al, 1984). Decreased plasma clearence of triacylglycerol as a result of diminished
activity
of
lipoprotein
lipase
has
been
reported
as
contributory
to
the
hypertriacylglycerolaemia induced by sepsis (Kaufmann et al , 1976; Robin et al ,
1981; Lanza-Jacoby et al , 1982).
Hepatic lipogenesis in vitro , measured by the incorporation of [114C]acetate into lipids in hepatocytes isolated from rats with pneumococcal infection,
is increased in response to sepsis (Canonico et al , 1977). There appears to be no
information, however, on rates of lipid synthesis in vivo in the liver or adipose tissue
after induction of sepsis.
Total cholesterol concentration in serum has been reported
to increase,
decrease, or remain unchanged during sepsis (Lindell et al , 1964; Fiser et al , 1971;
information on the dynamics involved in cholesterol synthesis, transport and
utilization. Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic
acid, showed increased incorporation of the labelled mevalonate into free cholesterol
in plasma when compared to control monkeys (Fiser et al , 1971). In vitro ,
[14C]acetate incorporation into cholesterol by isolated hepatocytes from rats with
pneumococcal infection was significantly increased as compared to the incorporation
by hepatocytes from control rats (Cannonico et al , 1977). However, there appears
to be no information on the rates of hepatic cholesterol synthesis in vivo in response
to sepsis.
Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated
rats (Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats
(Pace et al , 1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from
rats with pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the
flow of acetyl-CoA toward lipogenesis and cholesterolegenesis in vivo rather than to
ketogenesis or oxidation in the Krebs tricarboxylic acid cycle (Fig. 6.1.).
Fig. 6.1
To investigate the time course of changes in vivo in lipogenesis in the liver and white
and brown adipose tissue, and hepatic cholesterolegenesis, after the induction of
sepsis, caecal-ligated and punctured rats were studied. Sham-operated rats were
used as controls.
Experimental Design
Rats, after being subjected to caecal-ligation and puncture or shamoperation, were fasted for 12, 24 or 48 h before livers were removed and studied
(Fig.6.2.).
Fig. 6.2.
OP
12 h
24 h
48 h
Rats
3 H O injected
2
Livers removed
Lipid exctraction
Lipogenesis
Cholesterolegenesis
Microsomal preparation
HMGCoA reductase activity
For details on the operations and the techniques involved in the
measurement of lipogenesis, cholesterolegenesis, preparation of microsomes and
the measurement of hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and
lipoprotein lipase activities see Chapter 2. A group of rats had their bile duct ligated
immediately before either caecal-ligation and puncture or sham-operation was
performed (Chapter 2).
Results
The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo
was
significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48
h after the
operations, as compared to that of livers from sham-operated animals (Fig. 6.3.).
Fig. 6.3.
Rate of H
3
incorporation
O
into fatty acids
2
**
(7)
(4)
4.0
3.0
*
(18)
Sham-op
Septic
(3)
(13)
2.0
* P < 0.05
** P < 0.001
(6)
1.0
X + S.E.M.
0.0
12
24
48
found to be present in livers from septic rats when compared to those found in livers
from sham-operated rats (Fig. 6.4.) as early as at 12 h after the operation.
Fig. 6.4.
Rate of H3 incorporation
into non-saponifiable lipids
2O
*
(4)
7
6
Sham-op
Septic
**
(7)
5
**
(18)
4
3
(3)
* P < 0.05
(6)
** P < 0.001
2
(13)
X + S.E.M.
1
0
12
24
48
Therefore, livers from septic rats have significantly increased rates of total
lipogenesis
as compared to those of sham-operated rats. This increase in lipid
synthesis appears to occur both in rates of formation of saponifiable (fatty acids) and
non-saponifiable (squalene, cholesterol, other sterols) fractions (Fig. 6.5.).
Fig. 6.5.
Rate of H
10
3
incorporation
into lipids
2O
*
*
8
*
6
saponified
non-saponified
4
2
* P < 0.001
0
sham
1
septic
2
12 h
sham
septic
3
4
24 h
sham
septic
5
6
48 h
Effects of sepsis on squalene and cholesterol synthesis in vivo
and
hydroxymethylglutaryl-CoA reductase activity in the liver
The non-saponifiable fraction was separated into the various sterol fractions.
Sepsis produced a significant increase in the rate of squalene formation in the liver at
24 h after the operation (Fig. 6.6.). No change in rate was observed at the other time
points studied.
Fig. 6.6.
Rate of H
3
incorporation
into squalene
O
2
*
(7)
(4)
0.50
(18)
0.40
(3)
Sham-op
Septic
(13)
0.30
(6)
0.20
* P < 0.05
0.10
X + S.E.M.
0.00
12
24
48
Starvation caused the incorporation of 3H2O into cholesterol to decrease
with time in both sham-operated and caecal-ligated and punctured rats. However, the
rates of cholesterol synthesis in the livers of septic rats in vivo were significantly
higher than those found in the livers of sham-operated rats. This increase (2.3 fold)
occured as early as 12 h after the operations reaching a four-fold increase at 48 h
(Fig. 6.7.).
Fig. 6.7.
Rate of H
3incorporation
2O
into cholesterol
*
(4)
4.0
sham-op
septic
3.0
2.0
**
(11)
**
(18)
(3)
* P < 0.05
** P < 0.001
(11)
1.0
(12)
X + S.E.M.
0.0
12
24
48
livers of septic rats was significantly increased compared to that found in livers of
sham-operated rats at 48 h after the operations (Fig. 6.8.).
Fig. 6.8.
*
(7)
20
sham-op
septic
10
(4)
* P < 0.02
X + S.E.M.
0
48 h
HMG-CoA reductase is phosphorylated by a protein kinase. Phosphorylation
leads to inactivation of the enzyme. The activity of both the phosphorylated ('inactive'
form) and the non-phosphorylated or expressed form of this enzyme (Fig. 6.9.) was
measured in livers from septic and sham-operated rats at 48 h after the operation.
Sepsis induced no appreciable change in the expressed activity of HMG-CoA
reductase (non-phosphorylated or 'active'). As there was a significant increase in the
total enzyme activity (Fig. 6.8.) this was mainly due to an increase in activity of the
phosphorylated or 'inactive' form of the enzyme (Fig. 6.9.).
Fig. 6.9.
18
*
(7)
16
14
12
sham-op
septic
10
(7)
8
6
(4)
* P < 0.01
4
(4)
2
X + S.E.M.
0
'active'
phosphorilated
phosphorylated
Effects of sepsis on lipoprotein lipase activity and rates of lipogenesis in vivo in
adipose tissue
In order to obtain some information on the changes in lipid metabolism in
adipose tissue, and to compared to those found in the liver, rates of lipogenesis and
lipoprotein lipase activity were measured in white and brown adipose tissue of septic
and sham-operated rats. Sepis caused no appreciable change in the rate of total lipid
synthesis in white adipose tissue at 12 h after the operation, however, in contrast to
the findings in the liver, sepsis decreased the lipogenic rate at 48 h in adipose tissue
(Fig. 6.10.) when compared to that of sham-operated rats.
Fig. 6.10.
8
(8)
6
(µmol/g tissue/h)
3H2O incorporation into lipids
7
5
(10)
(7)
4
(11)
sham-op
septic
3
2
X + S.E.M.
1
0
12
48
The rate of lipogenesis in brown adipose tissue of septic rats was
significantly decreased when compared to that of sham-operated rats, at 12 h after
the operations. However, at 48 h, brown adipose tissue from septic rats synthesized
lipids at similar rates to those of sham-operated rats (Fig.6.11.).
Fig. 6.11.
(µmol/g of tissue/h)
3H2O incorporation into lipids
20
(7)
(10)
(8)
10
(12)
sham-op
septic
* P < 0.05
X + S.E.M.
0
12
48
Lipoprotein lipase activity was measured in adipose tissue from septic and
sham-operated rats at 48 h after the operations. Sepsis did not cause appreciable
change in lipoprotein lipase activity in white adipose tissue (septic : 0.302±0.035 ,
n=9 ; sham-operated : 0.253±0.048 , n=8 ; mean±SEM with number of observations
in parenthesis). However, this enzyme was significantly more active in brown
adipose tissue from septic rats at 48 h (septic : 1.37±0.26 , n=9 ; sham-operated :
0.61±0.16 , n=8 ; P < 0.05 - Student t-test).
accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver
in vivo
The entero-hepatic circulation of bile acids plays an essential role in the
absortion of cholesterol from the intestine and in the regulation of cholesterol
metabolism in the liver (for review see : Myant, 1981 and Gibbons et al , 1982). The
diversion of bile acids, or the complete interruption of the bile duct, lead to the
absence of bile acids in the intestine, which in turn, supresses the normal uptake of
cholesterol from the intestinal lumen. There is some evidence that it is the transport
of intestinal cholesterol to the liver which directly determines the rates of hepatic
cholesterol synthesis (Weis & Dietschy, 1975). This view is supported by the fact that
restoration of the entero-hepatic circulation, in rats subjected to biliary diversion,
failed to prevent the rise in hepatic cholesterolegenesis, whereas, the infusion of
chylomicron cholesterol lead to a prevention in the rise of hepatic sterol synthesis
rates (Gibbons et al , 1982). In addition, bile acids inhibit cholesterol 7Ï-hydroxylase
(the rate-limiting enzyme in the process of bile acids formation from cholesterol) and,
therefore, would prevent the catabolism and removal of cholesterol from the liver,
resulting in suppression of HMG-CoA reductase activity (Nervi & Dietschy, 1978).
The possibility of bile acids having a direct effect on HMG-CoA reductase, in the liver,
would suggest diversion rather than obstruction of the bile duct as the resonable
approach to study the metabolism of cholesterol in the liver. However, opposing the
idea of a direct bile acid effect is the fact that partial or complete biliary obstruction
cause an increase, rather than the expected decrease, in hepatic cholesterol
synthesis (Fredrickson et al , 1953; Gibbons et al , 1982). The absorption of bile
acids and cholesterol in the intestinal lumen of caecal-ligated and punctured rats may
be impaired due to the development of an adynamic ileus in these rats. Therefore,
we decided to produce a complete interruption of the entero-hepatic circulation of bile
acids, by ligating the bile duct of septic and sham-operated rats at 48 h after the
operation, to try to evaluate the contribution of the impairment in the absorption of
cholesterol and bile acids from the gut to the elevated rates of hepatic
cholesterogenesis found in this septic model (Fig. 6.7.).
The complete interruption of the entero-hepatic circulation of bile acids
obtained by bile duct ligation (BDL)
produced a significant increase in the rates of cholesterolegenesis in livers of shamoperated+BDL rats as compared to those of sham-operated rats at 48 h after the
operations. There was, however, no significant further increase in the rates of
cholesterol synthesis in the livers of septic rats+BDL as compared to caecal-ligated
and punctured rats (Fig. 6.12.).
Fig. 6.12.
Hepatic cholesterol synthesis and BDL
4
²
3
( µmol/g wet wt. liver/h)
3H2O incorporation into cholesterol
(4)
(8)
² P < 0.001
° sham-op/no BDL
(18)
2
sham-op
septic
1
(12)
X + S.E.M.
0
No 1BDL
BDL
2
There was no appreciable effect of BDL on the rate of fatty acid synthesis in shamoperated animals (sham-op+BDL : 1.95±0.13, n=4 ; sham-op : 1.95±0.24, n=13).
This stimulatory effect produced by bile-duct ligation was, therefore, specific for
cholesterol synthesis.
Discussion
Hypertriacylglycerolaemia is a comon finding during sepsis (Fig. 3.6. Chapter 3) and has been thought to be a consequence of both increased
triacylglycerol production
and decreased tryacylglycerol clearance from the
circulation (Robin et al , 1981). Hepatic esterification of [1-14C]oleate was increased
by this model of sepsis (Fig. 5.6. - Chapter 5) and this was accompanied by
unchanged lipoprotein lipase activity in white adipose tissue from septic rats at 48 h
after the operation. The rate of lipogenesis in white adipose tissue, however, was
decreased by sepsis (Fig. 6.10.) at the same time point.
Sepsis did not induce appreciable change in the rate of lipid synthesis in
brown adipose tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase
activity was, however, significantly increased in this tissue in septic rats, at the same
time point, suggesting the hydrolysed triacylglycerol is being oxidised and used for
energy or
heat production rather than for lipogenesis. Although high rates of
lipogenesis may be necessary for the production of sufficient amounts of nonesterified fatty acids for the activation of thermogenin (Cannon & Nedergaard, 1985),
thermogenesis can also occur without stimulated lipogenesis, the two being
regulated independently (Himms-Hagen, 1985).
Sepsis caused a significant increase in the rate of hepatic production of lipids
in vivo at all time points studied (Fig. 6.5.). There was increased production not only
of fatty acids, represented by the saponifiable lipid fraction (Fig. 6.3.) but also in the
total non-saponifiable lipid fraction (Fig. 6.4.).
During sepsis many factors create a large demand for cholesterol, including
increased hepatic production of lipoproteins (necessary to match with the increased
triacylglycerol synthesis) and possibly increased cell membrane synthesis at tissues
involved with defence and repair. As this need for cholesterol cannot be met from
dietary sources because the gut is not always available to absorb nutrients e.g.
during intra-abdominal sepsis, endogenous synthesis within the liver is expected to
increase. In fact, cholesterol synthesis was significantly increased in vivo in the
livers of septic rats as early as 12 h after the operation (Fig. 6.7.) while squalene
synthesis was increased only at 24 h (Fig. 6.6.).
Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.5. & 6.7.)
diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest
sepsis directs the flow of acetyl-CoA
preferentially toward lipid and cholesterol
synthesis rather than to partial oxidation with ketone body production or complete
oxidation in the tricarboxylic acid cycle.
As absorption of bile acids and cholesterol may be partially impaired in this
model of sepsis due to the presence of an adynamic ileus, in response to the
localized peritonitis, this may be a contributory factor to the raised cholesterol
synthesis in the livers of septic rats. Although hepatic fatty acid synthesis was not
affected by bile duct ligation (BDL) in sham-operated rats, at 48 h after the operation,
cholesterol synthesis rates in the livers of these rats were increased to levels
comparable to those of septic rats (Fig. 6.12.). However, rats subjected to
subcutaneous bacterial injection (Canonico et al , 1977), and therefore less likely to
develop an adynamic ileus, had increased rates of cholesterogenesis in vitro . This
finding does not support the view that partial interruption of the bile acid hepatoenteric circulation is the main reason for the increase in cholesterol synthesis in vivo
,found in this model of sepsis, and therefore favours an independent effect of sepsis.
Hydroxymethylglutaryl-CoA reductase activity and cholesterolegenesis were
measured in the same animals. Septic rats presented with higher total enzyme
activity (Fig. 6.9.) along with augmented cholesterol synthesis (Fig. 6.8.). There was ,
however , no change in the activity of the 'active' or expressed form of the enzyme
(Fig. 6.10.). Insulin, present in higher concentrations in the plasma of septic rats at all
time points studied (Fig. 3.5. - Chapter 3) may have exerted its longer-term effect by
increasing this enzyme synthesis rather than changing the activation state of the
enzyme.
The increased rates of hepatic lipogenesis induced by caecal-ligation and
puncture give support to the view of an inhibition in the entry of long-chain acyl-CoA
in the mitochondria of hepatocytes from septic rats by higher concentrations of
malonyl-CoA, an intermediate in the lipogenic pathway (see Chapter 5). This
stimulation in the rates of hepatic lipogenesis and cholesterolegenesis by sepsis may
well be due to greater availability of lactate (Figs. 3.2. & 3.9. - Chapter 3) and
pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the blood and liver, and the presence of
hyperinsulinaemia (Fig. 3.5. - Chapter 3).
CHAPTER 7
The effects of modulation of plasma insulin on hepatic metabolism during the
induction of sepsis
Introduction
The model of sepsis used in the present work caused substantial changes in
hepatic and peripheral metabolism. These included increased blood and hepatic
concentrations of gluconeogenic precursors, namely, lactate, pyruvate and alanine but no
change in glucose concentration (Chapter 3). There was also an impairment of the
hepatic capacity for gluconeogenesis and ketogenesis (Chapters 4 & 5) in vitro , and
stimulation of lipogenesis and cholesterogenesis
in vivo (Chapter 6). Thus despite being starved, the septic rats, in terms of hepatic
metabolism, behaved more like fed than fasted rats.
Caecal-ligated and punctured rats had higher plasma concentrations of insulin
at 12, 24 and 48 h after the operation (Fig. 3.5. - Chapter 3). Hyperinsulinaemia has also
been reported to occur in various experimental models of sepsis (Ryan et al , 1974;
Wichterman et al , 1979; Beisel & Wannemacher, 1980; Neufeld et al , 1980; Neufeld et
al , 1982; Clemens et al , 1984) and in the septic man (Shaw et al , 1985; Long et al ,
1985; Frayn, 1986). Higher concentrations of plasma insulin have also been reported
during endotoxaemia (Yelich & Filkins, 1980; Neufeld et al , 1982). Insulin is known to
stimulate lipogenesis and decrease gluconeogenesis and ketogenesis by its short-term
actions on the activation state of key enzymes (e.g. acetyl-CoA carboxylase) and by
longer-term regulation
of enzyme concentration (Denton et al , 1981). Therefore, the key factor which
prevented the septic rats from responding in the same way as the sham-operated rats to
starvation may be the raised plasma insulin. Although plasma glucagon is also increased
during sepsis (Beisel & Wannemacher, 1980; Shaw et al , 1985) its stimulatory effects
on ketogenesis and gluconeogenesis and its inhibitory effects on lipogenesis may be
antagonized by the elevated plasma insulin concentration.
A possible approach to investigate the postulated regulatory action of insulin,
upon the metabolic changes in sepsis, is to make the septic rats insulin-deficient.
Streptozotocin, an N-methylnitrosourea derivative, is a potent diabetogenic agent, being
extremely toxic to the ß-cell both in vitro and in vivo (Bolaffi et al , 1986). Its toxicity
for the ß-cells is linked to its capacity to accumulate rapidly in these cells (Gaulton et al ,
1985). Streptozotocin can supress insulin synthesis and secretion as early as 1 h after
exposure, this being followed by progressive necrosis and death of the ß-cell, in 1-3
days, resulting in permanent hyperglycaemia (for review see: Cooperstein & Watkins,
1981; Weiss, 1982). Neufeld et al (1980) reported a reversal of the hyperketonaemic
state induced by diabetes associated with infection (when total ketone body
concentration reached 12 µmol/ml of plasma) by insulin replacement (2 Units), in rats
previously (24 h) injected with streptozotocin (100mg/kg).
Another possibility to block insulin release in vivo in septic rats is the use of
mannoheptulose (Fig. 7.1.). This is a seven-carbon sugar, found in significant amounts in
avocado pears (for review see Simon & Kraicer, 1966 and Simon et al , 1972), which has
the ability to decrease insulin secretion by competitive inhibition of glucokinase and
hexokinase in the ß-cell (Coore & Randle, 1964; Malaisse et al , 1968).
Fig. 7.1.
Glucokinase is only present in the liver and pancreas and controls glucose
phosphorylation, the limiting-step of glycolysis. The metabolism of glucose to lactate
and pyruvate appears to be a very important signal for insulin release (Randle et al ,
1968), and as the capacity of the glycolytic pathway to handle glucose is decreased, by
the competitive action of mannoheptulose, so insulin release is decreased.
Therefore, to investigate the role of hyperinsulinaemia in the changes in
hepatic metabolism during the induction of sepsis, short-term decrease in plasma insulin,
by administration of mannoheptulose or streptozotocin, was induced in caecal-ligated
and punctured rats. The effects of mannoheptulose treatment in sham-operated rats was
also studied. To investigate whether the changes brought about by mannoheptulose
treatment of septic rats was due to the inhibition of insulin secretion the hormone was
administered with mannoheptulose to a group of septic rats, at 48 h after the induction of
sepsis.
Experimental design
Rats, after being subjected to caecal-ligation and puncture or sham-operation,
were fasted for 24 or 48 h when livers were removed (Fig. 7.2.) and blood collected.
Fig. 7.2.
Treatment to lower plasma insulin consisted of injection of either mannoheptulose
(500mg/200g body wt. - s.c.) or streptozotocin (50mg/kg body wt. - i.v.), 3 h before
removal of liver and 2 h before 3H2O injection (i.p.). To a group of septic rats, treated
with mannoheptulose, neutral insulin (Actrapid - 2 Units) was injected subcutaneously
15 min after the mannoheptulose injection (at 2 h and 45 min before removal of liver) at
48 h after the induction of sepsis. For details of the operations and the techniques
involved in the measurements of blood and hepatic metabolites, lipogenesis,
cholesterogenesis,
preparation
of
microsomes
and
the
measurement
of
hydroxymethylglutaryl-CoA (HMGCoA) reductase activity see Chapter 2.
Results
For purposes of comparison, the results of septic and sham-operated rats,
presented in Chapter 3 and Chapter 6, will also be included in the Figures of this chapter.
The effects of mannoheptulose or streptozotocin treatment on changes in concentrations
of blood glucose, lactate, pyruvate and alanine during the induction of sepsis
As already discussed sepsis caused no appreciable change in blood glucose
concentration at 24 and 48 h after the operation, but septic rats treated with either
mannoheptulose or streptozotocin became hyperglycaemic (Fig. 7.3.).
Fig. 7.3.
This could be the result of either increased hepatic glucose formation or
decreased glucose utilization in the periphery , or a combination of these factors.
Sepsis is known to cause hyperlactataemia. Septic rats had increased blood
lactate concentrations at 24 and 48 h after the operation. Mannoheptulose treatment
was unable to reverse the hyperlactataemia found in septic rats at either time points
(24 h and 48 h). When septic rats were treated with streptozotocin, lactate concentration
was not different from either septic or sham-operated rats at 48 h after the operation (Fig.
7.4.).
Fig. 7.4.
Blood pyruvate concentration was unchanged by sepsis at 24 h after the
operation. At 48 h septic rats
had significantly increased blood concentrations of
pyruvate as compared to those of sham-operated rats. Treatment of septic rats with
mannoheptulose reversed this increased blood pyruvate concentration induced by sepsis
at both 24 and 48 h after the operation. Septic rats treated with streptozotocin, however,
had higher concentrations of blood pyruvate at 48 h after the operation (Fig. 7.5.).
Fig. 7.5.
Blood alanine concentration was not appreciably changed by sepsis at 24 h
after the operation. However it was significantly elevated in caecal-ligated and punctured
rats as compared with sham-operated rats at 48 h after the operation. Mannoheptulose
injection in septic rats caused no change in blood alanine concentration at 24 h after the
operation. At 48 h , however, septic rats treated with mannoheptulose had significantly
decreased concentrations of blood alanine as compared to septic rats (Fig. 7.6.).
Fig. 7.6.
Although streptozotocin treatment tended to decrease the concentrations of blood alanine
in septic rats at 48 h after the operation, this change was not statistically significant (Fig.
7.6.). Mannoheptulose treatment, therefore, caused blood concentrations of pyruvate and
alanine to decrease and induced hyperglycaemia, whereas the only significant effect of
streptozotocin was hyperglycaemia.
The effects of mannoheptulose or streptozotocin treatment on the concentrations of
blood ketone bodies during the induction of sepsis
Sepsis is known to cause inhibition of the hyperketonaemia in response to
starvation, as confirmed by the lower blood concentrations of ketone bodies at 24 and 48
h after the operation (Fig. 7.7.). Treatment to lower plasma insulin (mannoheptulose or
streptozotocin injection) in septic rats returned the blood ketone body concentrations to
the values of the sham-operated rats (Fig. 7.7.)
Fig. 7.7.
The effects of mannoheptulose or streptozotocin treatment on plasma insulin
concentration during the induction of sepsis
Sepsis caused a significant increase in plasma insulin concentration at 24 and
48 h after the operation. When mannoheptulose was administered to septic rats, at 24 h
after the operation, plasma insulin concentrations were lower but not significantly
different from those of caecal-ligated and punctured rats. At 48 h after the operation
mannoheptulose treatment caused a significant decrease in plasma insulin concentration,
as compared to that of septic rats, but these values were still significantly higher than
those of sham-operated rats. Septic rats injected with streptozotocin had a significant
decrease in insulin concentrations in plasma, as compared to those of untreated septic
rats, at 48 h after the operation (Fig. 7.8.).
Fig. 7.8.
The effects of mannoheptulose or streptozotocin treatment on hepatic concentrations of
lactate, pyruvate, alanine and glucose during the induction of sepsis
Hepatic concentrations of lactate were significantly increased by sepsis at 24
and 48 h after the operation. The administration of mannoheptulose or streptozotocin
treatment did not cause an appreciable change in the hepatic concentrations of lactate
(Fig. 7.9.).
Fig. 7.9.
Hepatic lactate concentration
**
(3)
3
µmol/g fresh wt. liver
*
(5)
2
1
**
(6)
**
(12)
*
(5)
(6)
(9)
Sham-op
Septic
Septic+MMannohept.
Septic+Strz
Streptoz.
* P < 0.05
** P < 0.001
versus Sham-op
X + S.E.M.
0
2424
48
The hepatic concentrations of pyruvate were also increased during induction
of sepsis at 24 and 48 h after the operation. Mannoheptulose injection in septic rats,
however, caused a significant decrease in the concentrations of pyruvate in the liver at 48
h after the operation (Fig. 7.10.). The administration of streptozotocin caused no change
on the hepatic concentrations of pyruvate in septic rats (Fig. 7.10.).
Fig. 7.10.
Sepsis caused an increase in hepatic concentrations of alanine at 48 h after the
operation. Mannoheptulose treatment did not have a significant effect on the hepatic
concentrations of alanine in septic rats at 24 or 48 h after the operation. Concentrations
of alanine in livers from septic rats treated with streptozotocin were not different from
those of either septic or sham-operated rats at 48 h after the operation (Fig. 7.11.).
Fig. 7.11.
Hepatic glucose concentration remained unchanged by sepsis at 24 and 48 h
after the operation. However, septic rats treated with mannoheptulose or streptozotocin
had a significant increase in glucose concentrations in the liver (Fig. 7.12.).
Fig. 7.12.
The effects of mannoheptulose or streptozotocin treatment on the hepatic concentration
of metabolites involved in gluconeogenesis during the induction of sepsis
The results of the effects of either mannoheptulose or streptozotocin
administration to caecal-ligated and punctured rats on the hepatic concentration of
metabolites involved in the glucose synthesis pathway, at 24 and 48 h after the operation,
are presented in Table 7.1..
The effects of mannoheptulose or streptozotocin treatment on the hepatic
concentration of metabolites involved in gluconeogenesis after the induction of
Table 7.1.
sepsis
in vivo
____________________________________________________________
For full details see Chapter 2. Results are mean values ± S.E.M. with the number of observations in
parentheses. Significance of difference by Student's t-test from septic rats:
* P< 0.05; ** P < 0.01. The
following abbreviations are used: Septic+M - Septic + Mannoheptulose; Septic+Strz - Septic +
Streptozotocin;
PEP
Phosphoglycerate;
-
Phosphoenolpyruvate;
2PGA
-
D-2-Phosphoglycerate;
3PGA
-
D-3-
DHAP - Dihydroxyacetone phosphate; FDP - Fructose-1,6-bisphosphate; G-6-P -
Glucose-6-phosphate.
____________________________________________________________
Metabolite
Concentration (µmol/g fresh wt. of liver)
__________________________________________________________
24 h
_______________________
Septic
Septic+M
48 h
____________________________________
Septic
Septic+M
Septic+ Strz
_______________________________________________________________________
Alanine
1.51±0.28(5)
1.09±0.10(6)
1.24±0.16(11)
1.10±0.25(5)
1.47±0.13(6)
1.21±0.19(14)
1.46±0.34(5)
2.07±0.61(4)
Pyruvate 0.093±0.014(5) 0.066±0.015(6)
0.078±0.009(9)
0.046±0.01(5)*
0.095±0.019(4)
PEP
0.28±0.03(6)
1.18±0.68(3)
Lactate
1.66±0.42(5)
0.32±0.02(5)
0.27±0.03(5)
2PGA
0.28±0.05(9)
0.22±0.03(4)
0.076±0.005(5)
0.065±0.004(6)
0.068±0.005(9)
0.075±0.005(5)
0.67±0.03(6)
0.73±0.06(9)
0.009±0.003(5)
0.015±0.002(6)
0.014±0.002(9)
0.014±0.004(5)
0.013±0.003(5)
0.015±0.004(6)
0.019±0.003(9)
0.018±0.003(5)
0.24±0.03(5)
0.27±0.02(6)
0.17±0.03(9)
0.068±0.007(4)
3PGA
0.73±0.05(5)
0.71±0.06(5)
0.76±0.08(4)
DHAP
0.012±0.002(4)
FDP
0.024±0.003(4)
G-6-P
0.22±0.07(5)
0.090±0.025(4)*
Glucose
5.97±0.56(6)
12.02±0.96(6) ** 6.03±0.20(8)
10.69±1.08(5)**
7.62±0.32(4)*
_______________________________________________________________________
The same results are presented as percentage of hepatic metabolites in septic rats and
caecal-ligated rats subjected to mannoheptulose or streptozotocin treatment, in relation
to sham-operated metabolite concentrations, when the latter are set at 100% (Figs.
7.13.,7.14. & 7.15.).
Sepsis caused a significant increase in the hepatic concentrations of lactate,
pyruvate and glucose 6-phosphate at 24 h after the operation. When septic rats were
treated with mannoheptulose the hepatic pyruvate concentration was no longer
significantly increased, compared to the concentrations found in the livers of shamoperated rats. Mannoheptulose injection caused no
appreciable change in the high concentrations of hepatic glucose
6-phosphate, but caused a significant increase in hepatic glucose concentration (Fig.
7.13.)
Fig. 7.13.
At 48 h after the operation, sepsis caused increases in the concentrations of most
metabolites involved in gluconeogenesis, with the exception of D-2-phosphoglycerate
and dihydroxyacetone phosphate and glucose. Septic rats, treated with mannoheptulose,
showed a significant decrease in hepatic pyruvate concentration and a significant
increase in hepatic glucose concentration (Fig. 7.14.).
Fig. 7.14.
Septic rats had increased hepatic concentrations of glucose 6-phosphate as
early as 24 h after the operation, as compared to sham-operated rats (Table 3.1. - Chapter
3; Fig. 7.13.). However, when streptozotocin was administered to lower plasma insulin
in septic rats, at 48 h after the operation, there was a significant decrease in the hepatic
concentration of glucose-6-phosphate, as opposed to a significant elevation in hepatic
glucose concentration. Glucose 6-phosphatase activity is known to be increased in livers
of diabetic animals (Ashmore & Weber, 1959; Garfield & Cardell, 1979). If short-term
treatment with streptozotocin causes a similar change in glucose 6-phosphatase activity,
this may explain the decrease in glucose 6-phosphate leading to increased glucose
concentration in the liver of septic-treated rats (Fig. 7.15.)
Fig. 7.15.
The pattern of metabolite changes in septic rats treated with mannoheptulose,
at 24 and 48 h after the operation, suggests an increase in glucose synthesis at the
expense of augmented pyruvate
utilization. As the hepatic cytosolic redox state (NAD+/NADH ratio) is in equilibrium
with the concentrations of lactate/pyruvate, the fall in hepatic pyruvate concentration
during mannoheptulose treatment,
in septic rats, may also reflect a decrease in the NAD+/NADH ratio, which in turn could
lead to a stimulation of gluconeogenesis (Williamson et al , 1967). The diminished
hepatic concentration of glucose-6-phosphate associated with an increase in hepatic
glucose concentration, in septic rats treated with streptozotocin, suggests increased flux
through the glucose 6-phosphatase step.
The effects of mannoheptulose or streptozotocin treatment on the changes in hepatic
concentrations of ketone bodies, acetyl-CoA and ATP during the induction of sepsis
The inhibition of the hyperketonaemia in response to starvation caused by
sepsis was reversed by mannoheptulose or streptoztocin (Fig. 7.7.). Ketone body
concentrations in the liver were also decreased by sepsis at 48 h after the operation.
Injection of mannoheptulose or streptozotocin, in septic rats, caused an elevation in the
hepatic concentrations of ketone bodies to similar concentrations as those found in the
livers of sham-operated rats at 48 h after the operation (Fig. 7.16.)
Fig. 7.16.
Sepsis or sepsis accompanied by mannoheptulose treatment did not cause
appreciable change in hepatic concentration of acetyl-CoA at 24 h after the operation as
compared to those of sham-operated rats. Acetyl-CoA concentrations, however,
decreased significantly in the livers of septic rats as compared to those of sham-operated
rats at 48 h after the operation. Treatment to lower
plasma insulin (mannoheptulose or streptozotocin injection) decreased even further the
hepatic concentration of acetyl-CoA to values significantly lower than those of septic
animals (Fig. 7.17.).
Fig. 7.17.
ATP concentrations in the livers of septic rats were not significantly different
from those of sham-operated animals at 24 and 48 h after the operation. Mannoheptulose
treatment of septic rats did not cause appreciable change in hepatic ATP concentration at
24 or 48 h after the operation (Fig 7.18).
Fig. 7.18.
Administration of streptozotocin to septic rats caused a slight decrease in hepatic ATP
concentrations,
to values significantly lower than those of sham-operated rats
(Fig.7.18.).
The effects of mannoheptulose or streptozotocin treatment on the changes in rates of
lipogenesis and cholesterogenesis in vivo during the induction of sepsis
Sepsis stimulated the rates of fatty acid (saponifiable lipid fraction) synthesis
at 24 and 48 h after the operation. Mannoheptulose treatment, given to septic rats at 24
and 48 h after the operation, caused a significant reduction in the rates of incorporation
of 3H2O into fatty acids, as compared to those of caecal-ligated and punctured rats (Fig.
7.19.). The rates of tritiated water incorporation into fatty acids was lower, but did not
reach statistical significance, in septic rats injected with streptozotocin at 48 h after the
operation, as compared to those of caecal-ligated and punctured rats (Fig. 7.19.).
Fig. 7.19.
Increased rates of synthesis of total non-saponifiable lipids in vivo were found
in livers of septic rats at 24 and 48 h after the operation. Administration of
mannoheptulose or streptozotocin to septic rats caused a decrease in the rates of 3H2O
incorporation into non-saponifiable lipids (squalene, cholesterol and other steroids) at 24
and 48 h after the induction of sepsis (Fig. 7.20.).
Fig. 7.20.
The non-saponifiable fraction was separated into squalene, cholesterol and
other sterol fractions. The rates of 3H2O incorporation into squalene were increased, at
24 h after the operation, in livers from septic rats. Mannoheptulose treatment, given to
septic rats, at 24 h after the operation tended to decrease 3H2O incorporation into
squalene, as compared to that of septic rats, but this was not statistically significant (Fig.
7.21). When mannoheptulose or streptozotocin were injected in septic rats, 48 h after the
operation, there was a significant decrease in the rates of 3H2O incorporated into
squalene, when compared to those of septic rats (Fig. 7.21.).
Fig. 7.21.
The rates of cholesterogenesis in vivo
in the livers of septic rats were
significantly increased at 24 and 48 h after the operation. Treatment of septic rats with
mannoheptulose or streptozotocin caused a significant decrease in the rates of 3H2O
incorporation into hepatic cholesterol in vivo , as compared to those of septic rats, at 24
and 48 h after the operation (Fig. 7.22.).
Fig. 7.22.
The
reduction
of
3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA)
to
mevalonate by HMG-CoA reductase plays a key role in the regulation of the rate of
cholesterol synthesis. Phosphorylation of HMG-CoA reductase leads to inactivation
whereas the non-phosphorylated (expressed) form is the 'active' form of the enzyme
(Gibson & Ingebritsen, 1978; Denton et al , 1981; Gibson & Parker, 1987). Sepsis did
not cause appreciable change in the activity of the expressed or 'active' form of the
enzyme, but increased significantly the phosphorylated form of HMG-CoA reductase, at
48 h after the operation (Fig. 7.23.).
Fig. 7.23.
Administration of mannoheptulose or streptozotocin to septic rats at 48 h after the
operation, had no effect on this pattern of changes induced by sepsis, either in the
activity of the expressed or active form or in the activity of the phosphorylated form of
this enzyme, which remained higher as compared to that of sham-operated rats.
There was a lack of correlation between the activity of HMG-CoA
reductase (unchanged), and cholesterol synthesis (decreased) in septic rats subjected to
mannoheptulose or streptozotocin treatment, as judged by the two-fold increase in the
ratio of total HMGCoA reductase activity/cholesterol synthesis in septic treated rats
(Septic: 10.4; Septic+Mannoheptulose: 18.8; Septic+Streptozotocin: 24.2).
The effects of mannoheptulose treatment on the metabolic changes of sham-operated rats
Sham-operated rats were treated with mannoheptulose in order to evaluate the
effects of this compound in the control situation. Table 7.2. summarizes the changes
brought about by mannoheptulose treatment on sham-operated rats, at 24 and 48 h after
the operation. For purposes of comparison, the data on sham-operated rats are also
included in the table.
Plasma insulin concentrations in sham-operated rats, treated with
mannoheptulose, were not significantly different from those of sham-operated rats
(Sham-op: 9.90±1.90 (n=10) versus 13.55±2.64 (n=4) - µU/ml plasma) at 48 h after the
operation. Despite plasma insulin concentrations remaining unchanged with the
mannoheptulose treatment, the metabolic changes found in the treated sham-operated
rats were compatible with those to be found under the influence of low insulin
concentration, namely, increased blood and hepatic concentrations of glucose and ketone
bodies. The sham-operated rats were hyperglycaemic in the presence of lower plasma
insulin concentrations, as compared to those of septic rats (Fig. 7.8.). This suggests that
both sham-operated and rats treated with mannoheptulose had a plasma insulin
concentration which was inappropriate for the blood glucose. The increase in hepatic
ketone bodies was accompanied by either unchanged (24 h) or increased hepatic acetylCoA concentration.
Table 7.2.
The effects of mannoheptulose treatment on the metabolic changes of
sham-operated rats in vivo
____________________________________________________________
For full details see Chapter 2. Results are mean values ± S.E.M. with the number of observations in
parentheses. Significance of difference by Student's t-test from septic rats: * P< 0.05; ** P < 0.01; *** P <
0.001. The following abbreviations are used: Sham-op - Sham-operated; Sham-op+M - Sham-operated +
Mannoheptulose;
PEP - Phosphoenolpyruvate; 2PGA - D-2-Phosphoglycerate; 3PGA - D-3-
Phosphoglycerate;
DHAP - Dihydroxyacetone phosphate; FDP - Fructose-1,6-bisphosphate; G-6-P -
Glucose-6-phosphate.
____________________________________________________________
Metabolite
_____________________________________________________
24
h
48 h
____________________________
Sham-op
_____________________________
Sham-op+M
Sham-op
Sham-op+M
_______________________________________________________________________
Blood
Lactate
1.56±0.19(9)
1.20±0.13(4)
1.14±0.14(9)
1.41±0.089(4)
Pyruvate
0.058±0.009(3)
0.040±0.007(3)
0.057±0.010(7)
0.044±0.012(4)
Alanine
0.24±0.015(9)
0.16±0.016(4)**
0.14±0.020(6)
0.16±0.014(4)
Glucose
4.27±0.32(9)
9.51±0.29(4)***
4.10±0.26(8)
8.97±0.95(4)***
Ketone bodies
0.79±0.13(9)
1.46±0.17(4)*
1.08±0.14(11)
2.02±0.33(4)**
Hepatic
Lactate
0.63±0.095(6)
0.28±0.080(4) *
0.36±0.064(11)
0.56±0.068(4)
Pyruvate
0.036±0.004(6)
0.029±0.008(4)
0.038±0.007(8)
0.043±0.002(3)
Alanine
0.94±0.19(6)
0.25±0.012(4)*
0.39±0.074(10)
0.043±0.062(4)*
PEP
0.26±0.052(6)
0.15±0.093(4)
0.14±0.032(8)
0.077±0.002(4)
2PGA
0.059±0.007(6)
0.045±0.050(4)
0.050±0.007(8)
0.022±0.011(4)
3PGA
0.67±0.14(6)
0.38±0.069(4)
0.43±0.075(8)
0.011±0.002(6)
0.010±0.006(4)
DHAP
0.010±0.003(8)
0.15±0.010(4)*
0.004±0.002(3)
FDP
0.012±0.003(6)
0.016±0.007(4)
0.007±0.001(8)
0.009±0.002(3)
G6P
0.093±0.037(6)
0.16±0.025(4)
0.075±0.017(8)
0.074±0.015(4)
6.19±0.55(6)
12.24±0.81(4)**
Glucose
5.66±0.32(7)
13.39±1.50(4)***
Ketone bodies
0.73±0.089(6)
1.68±0.14(4)***
1.65±0.14(12)
1.99±0.35(4)*
Acetyl-CoA
0.088±0.015(6)
0.053±0.016(4)
0.100±0.008(4)
0.189±0.023 (4)*
2.35±0.29(6)
1.66±0.15(4)
2.11±0.074(8)
2.88±0.51(4)
ATP
_______________________________________________________________________
In sham-operated rats hepatic lipogenesis was very low, and unaffected by
the mannoheptulose treatment (Fig. 7.24.)
Fig. 7.24.
Cholesterogenesis, on the other hand, was significantly decreased by
mannoheptulose treatment at 24 and 48 h after sham- operation (Fig.7.25.).
Fig. 7.25.
The effects of insulin replacement given to septic rats treated with mannoheptulose on
the metabolic changes induced by sepsis
To investigate whether the metabolite changes observed in septic rats treated
with mannoheptulose, were likely to be due to insulin deficiency, insulin was
administered to septic rats (48 h) treated with mannoheptulose. However, it must be
emphasised, that a pharmacological dose of insulin was used, and therefore, the resulting
hyperinsulinaemia was greater than that found in septic rats before the treatment with
mannoheptulose.
A summary of the results of insulin replacement in septic rats treated with
mannoheptulose is shown on Table 7.3.. For comparison, results obtained from
septic rats and septic rats treated with mannoheptulose are also included in the table.
Insulin
replacement
to
septic
rats
previously
treated
with
mannoheptulose caused profound hypoglycaemia associated with a decrease in
hepatic glucose concentration. Ketone body concentration in blood and liver also
decreased. The replacement with pharmacological doses of insulin was,
however, unable to increase the rates of either lipogenesis or cholesterogenesis
to levels comparable to those in septic rats.
Table 7.3.
The effects of insulin administration on the metabolic changes of septic
rats treated with mannoheptulose in vivo at 48 h after the operation
____________________________________________________________
For full details see Material and Methods (Chapter 2). Results are mean values ± S.E.M. with the number of
observations in parentheses. Significance of difference by Student's t-test from septic rats :
** P < 0.01 .
* P< 0.05;
From septic rats treated with mannoheptulose: ∆ P < 0.05; ∆∆ P < 0.01. The following
abbreviation is used: Septic+M - Septic + Mannoheptulose.
____________________________________________________________
Metabolite
_________________________________________________
Septic
Septic+M
Septic+M+Insulin
_______________________________________________________________________
Plasma
Insulin
35.83±3.88(10)
24.71±3.42(9)*
409.9±117.2(4)**∆∆
Blood
Lactate
2.37±0.22(12)
2.19±0.41(5)
0.120±0.019(10)
0.53±0.0.19(5)*
0.34±0.076(7)
0.19±0.017(5)*
3.90±0.11(9)
7.34±0.81(5)*
1.29±0.55(4)*
Pyruvate
0.076±0.027(4)
Alanine
0.17±0.049(4)
Glucose
0.54±0.061(4)**∆∆
Ketone bodies
0.59±0.055(14)
1.60±0.086(5)**
1.21±0.19(14)
1.46±0.34(5)
0.078±0.009(9)
0.046±0.010(4)*
0.37±0.027(4) ∆∆
Hepatic
Lactate
0.78±0.20(4)
Pyruvate
0.099±0.013 (4) ∆
Alanine
1.24±0.17(11)
1.10±0.25(5)
6.03±0.20(8)
10.69±1.08(5)*
0.55±0.25(4)
Glucose
1.54±0.14(4)**∆∆
Ketone bodies
0.78±0.052(14)
1.72±0.19(5)**
0.53±0.069(4) *∆∆
Hepatic
Lipogenesis
3.04±0.41(18)
1.61±0.15(14)**
1.48±0.13(4)*
Cholesterogenesis
1.73±0.22(18)
0.94±0.15(14)*
0.87±0.18(4) *
_______________________________________________________________________
Discussion
The aim of these experiments was to see whether lowering the raised plasma
insulin in septic rats, by treatment with mannoheptulose or streptozotocin, would return
the metabolic profile to that seen in the sham- operated animals. The treatment did
lower plasma insulin (Fig. 7.8.), particularly at 48 h after the operation. It must, however,
be emphasized that the plasma insulin was not returned to sham-operated values. A
summary of the significant changes brought about by mannoheptulose or streptozotocin
treatment of septic rats (48 h after the operation) are listed in Fig. 7.26.
Fig. 7.26.
The decrease in plasma insulin, after administration of mannoheptulose or
streptozotocin
to
septic
rats,
resulted
in
the
expected
metabolic
changes
(hyperglycaemia, hyperketonaemia and decreased lipogenesis). This finding supports the
view that it is the raised plasma insulin which is, in part, responsible for the anomalous
response of septic rats to starvation.
The fact that the hepatic glucose concentration was increased, by giving
septic rats mannoheptulose treatment, may reflect increased hepatic glucose synthesis.
The development of hyperglycaemia may be the result of either increased hepatic
synthesis or decreased glucose utilization by peripheral tissues. However, insulin
stimulated glucose uptake in vitro to the same extent in muscles from rats subjected to
caecal ligation and double puncture (needle gauge-18) or sham-operated rats, at 16 and
24 h after the operation (Wichterman et al , 1979).
The high lactate concentrations in the blood and in the liver of septic rats
remained unchanged after mannoheptulose or streptozotocin treatment, supporting the
view of continued lactate availability from glycolysis in the periphery. The lower
concentrations of pyruvate in the blood and liver of septic rats, subjected to
mannoheptulose injection, may be the result of increased hepatic pyruvate utilization or a
change in the cytosolic NADH/NAD ratio (Williamson et al , 1967). Increased
utilization of pyruvate can occur to meet the demand for the stimulated hepatic glucose
production, as indicated by the elevated hepatic glucose concentrations.
The pattern of changes in the concentrations of metabolites involved in
gluconeogenesis suggests that one effect of mannoheptulose is to increase glucose
synthesis, with the consequent decrease in hepatic pyruvate concentration, and an
increase in glucose concentration in the liver.
Streptozotocin treatment of caecal-ligated and punctured rats, on the other
hand, caused a decrease in the hepatic concentration of glucose-6-phosphate together
with an elevation in the concentration of glucose in the liver, suggesting increased
glucose synthesis from this immediate precursor. It must, however, be emphasised that as
the concentrations of plasma glucose and hepatic glucose rapidly equilibrate, any
increase in the latter may be due to a decrease in peripheral utilization. Streptozotocin
treatment, therefore, may have overcome the lower activity of glucose 6-phosphatase
found during sepsis (Canonico et al ; 1977). This may have been achieved if the effect
of short-term streptozotocin treatment increases the activity of glucose 6-phosphatase, as
it does in streptozotocin induced diabetic rats (Garfield & Cardell, 1979). Therefore, in
septic rats treated to lower plasma insulin the glucose 6-phosphate/glucose cycle appears
to operate preferentially towards glucose formation (Fig. 7.27.).
In contrast, Wannemacher et al (1980) measuring 14C-alanine incorporation
into glucose in vivo , in rats pre-treated with mannoheptulose and injected with
Streptoccocus pneumoniae (104) showed no increase in 14C incorporation into glucose,
as compared to controls. In addition, rats receiving 107 Streptoccocus pneumoniae had a
significantly lower incorporation of 14C into glucose, when compared to heat-treated
controls. Therefore, the milder bacterial treatment caused no appreciable change in
gluconeogenic rates in vivo with mannoheptulose treatment, whereas with the more
severe bacterial injection gluconeogenesis in vivo was decreased in mannoheptulose
treated rats. Insulin deficiency, with mannoheptulose treatment, was therefore not able to
help to overcome the decreased gluconeogenic rates found in very severe sepsis. These
results illustrate the importance of the severity of infection in the modulaton of the
metabolic response to sepsis.
Fig. 7.27.
The
injection
of mannoheptulose or streptozotocin increased the
concentration of blood and hepatic ketone bodies during sepsis. Treated septic rats
(mannoheptulose or streptozotocin), had their ketone bodies increased to concentrations
similar to those found in sham-operated rats. This suggests the
impairment in the utilization of long-chain fatty acids, in the livers of septic rats (see
Chapter 6), was overcome by lowering plasma insulin concentration. Mannoheptulose or
streptozotocin treatment had also a significant effect in decreasing the rates of
lipogenesis, and presumably lowering the concentration of malonyl-CoA. The lower
concentrations of malonyl-CoA would, in turn, activate carnitine acyl-transferase I
resulting in increased flux of long-acyl CoA into the mitochondria for ß-oxidation
leading to formation of acetyl-CoA, the immediate precursor for ketogenesis.
Treatment to lower plasma insulin in septic rats, caused a further decrease in
the hepatic concentration of acetyl-CoA. As there appears to be increased flux of acylCoA into the mitochondria for ß-oxidation leading to increased formation of acetyl-CoA,
the lower hepatic concentrations of acetyl-CoA could be the result of either increased
total oxidation in the Krebs cycle, partial oxidation to yield ketone bodies, or a
combination of both. However during long-term insulin deprivation (diabetes) or in
starvation, when ketogenesis is increased, hepatic concentrations of acetyl-CoA are
increased (Fig. 3.8. - Chapter 3). Lowering plasma insulin during a shorter period of time
(3h), in the sham-operated rats treated with mannoheptulose, caused an increase in
hepatic acetyl-CoA at 48 h after the operation (Table 7.2.). Sepsis, therefore, appears to
have an independent effect on decreasing the hepatic acetyl-CoA concentration, as
opposed to the effect of starvation (increase) or trauma and starvation accompanied by
mannoheptulose treatment (increase), the latter represented by the increase in acetylCoA in sham-operated rats treated with mannoheptulose (Table 7.2.). The reason for this
difference is not clear.
The ATP concentration in the livers of septic or sham-operated rats was not
significantly affected by the administration of mannoheptulose, whereas there was a
slight decrease in ATP concentration in septic rats treated with streptozotocin, as
compared to that found in the livers of sham-operated rats at 48 h after the operation.
The rates of incorporation of 3H2O into fatty acids and non-saponifiable lipids
were significantly increased by sepsis. Treatment to lower plasma insulin in septic rats,
decreased these rates, and livers from septic rats injected with mannoheptulose or
streptozotocin, produced fatty acids, and non-saponifiable lipids at significantly lower
rates when compared to those of septic rats; some of these rates were very similar to
those of sham-operated rats.
Sepsis caused a significant increase in the rates of cholesterogenesis (12, 24
and 48 h after the operation) and squalene formation (24 h after the operation).
Mannoheptulose or streptozotocin treatment also caused a decrease in the rates of
cholesterol and squalene synthesis in the liver, measured by the decreased incorporation
of 3H2O into these sterols.
To evaluate the regulation of cholesterol metabolism under these conditions,
the total (phosphorylated) and the expressed or 'active' form of HMG-CoA reductase
were measured in septic rats subjected to treatment to lower plasma insulin. There was a
lack of correlation between enzyme activity, which remained high, at rates comparable to
those of septic rats, and the decreased rate of cholesterol synthesis. The higher activity of
HMG-CoA reductase in septic rats at 48 h after the operation suggests higher plasma
insulin concentration may have had its longer-term action by increasing enzyme
concentration in the liver. During the short-term mannoheptulose or streptozotocin
treatment, insulin appeared not to exert its action by promoting covalent phosphorylation
-dephosphorylation leading to changes in the activation state of the enzyme, because the
activity of the expressed form of the enzyme remained unchanged by sepsis associated
with either mannoheptulose or streptozotocin injection. Easom & Zammit (1984)
developed a technique for rapid sampling of rat liver, by injecting ice-cooled sucrose
buffer into the portal system to try to clear the liver from red cells, and to provide
immediate ice-cooling of the organ. By using this technique they claim the
phosphorylation state of HMG-CoA reductase in vivo is better maintained in the
microsomal preparation for determination of enzyme activity. We have not used this
technique and, therefore, a portion of the expressed form may have been lost, but it is
likely that this loss was similar in sham-operated and septic rats. In addition, the changes
induced by sepsis were mainly in the total form of the enzyme (Fig. 7.25.).
The treatment to lower plasma insulin, in septic rats, therefore, was able to
promote the reversal of the changes in ketone body concentration in blood and liver, in
pyruvate concentration in liver and blood, in hepatic glucose-6-phosphate concentration ,
and to induce an increase in hepatic and blood glucose. Reversal of the rates of
cholesterogenesis, squalene synthesis and lipogenesis were also obtained. The
administration of insulin, in pharmacological dose, to septic rats treated with
mannoheptulose (Table 7.3.), caused a partial return of the metabolic profile of septic
mannoheptulose-treated rats to that found in septic rats.
The present results, therefore, support the view that the higher insulin
concentration is involved in the development of the changes which occur during the
induction of sepsis. Consequently, in many respects, septic rats subjected to
mannoheptulose or streptozotocin treatment, behaved more like sham-operated rats than
septic rats.
CHAPTER 8
General Discussion
The metabolic response to infection is part of a concerted physiological and
immunological response, which is dependent on a number of factors, including the
nutritional status, the hormonal milieu, and the stage and degree or severity of sepsis. The
interrelation between these factors is reflected in the large spectrum of the metabolic
changes observed in septic patients (Cerra, 1982; Wilmore, 1983; Forse & Kinney, 1985).
The severity of infection plays an important part in the septic process and can
modulate the metabolic changes induced by sepsis, as illustrated in Fig. 8.1. The
experimental model used in the work presented in this Thesis induced a moderate form of
sepsis, as compared with that of others (Chaudry et al , 1979; Wichterman et al , 1980).
The septic rats did not become hypothermic nor shocked. The blood lactate in the septic
group was increased as early as 12 h after the operation but never reached values defined
as those for lactic acidosis (5 mmol/l). They did not go through the late hypodynamic late
septic state (Chaudry et al , 1979), demonstrated by the fact that they did not develop
hypoglycaemia or hypoinsulinaemia at any time point studied.
Fig. 8.1.
The regulation of the metabolic response to sepsis
The exact cascade of events which leads to the establishment and evolution of the
septic response is starting to become clearer. Immunological, inflammatory and endocrine
mediators appear to act in concert to mount the metabolic and physiological response,
which appears to be fairly uniform and partially independent of the infective agent (Wiles
et al , 1980; Frayn, 1986).
In Chapter 1, various "mediators" of the response to sepsis were discussed,
including interleukin-1 or its sub-products (Dinarello & Wolff, 1982; Takemura & Werb,
1984), and cachectin (Tracey et al , 1986; Beutler & Cerami, 1987). These "mediators"
may represent the link between the immunological and the metabolic response. In
support of this view is the action of interleukin-1 (leucocytic endogenous mediator;
proteolysis inducing factor) in promoting the muscle catabolism and stimulating the ß-cells
to secrete insulin during sepsis (George et al , 1977; Clowes et al , 1983 and 1985).
The elevation in plasma insulin appears to be a key factor in the hepatic metabolic
changes induced by caecal-ligation and puncture in the rat. This led to the proposal of a
unified hypothesis for the regulation of the changes in hepatic metabolism by the action of
the raised plasma insulin (Chapter 7). Furthermore, treatment to lower plasma insulin
reversed many of the metabolic changes induced by sepsis, giving support to the important
modulatory role of insulin (Chapter 7). Whether the leucocyte-derived "mediators" also
play a direct role in the regulation of the metabolic response to sepsis, in the liver, requires
further investigation.
Recent evidence suggests that interleukin-1 injected into the rat in vivo , has a
stimulatory effect on hepatic metabolism in isolated hepatocytes (Roh et al , 1986). The
metabolic changes induced by interleukin-1 adminstration had a time-dependent pattern
and included an increase in glucose synthesis from alanine, stimulation of protein synthesis
and an increase in oxygen consumption. Interleukin-1 appears to exert its effect on the
liver indirectly, for no action was observed when interleukin-1 was added to the
hepatocytes' incubation medium. The infusion of cachectin into rats caused some of the
metabolic changes found during sepsis, such as hyperglycaemia and hyperlactataemia and
led to the development of changes in target organs similar to those found in the multiple
organ failure syndrome (Beutler & Cerami, 1987). Nevertheless, the regulatory role of
cachectin upon the metabolic changes in the liver still needs clarification. Further research
involving in vivo turnover studies might help to elucidate the action of these leucocytederived 'mediators'. The administration for example of monoclonal antibodies, anticachectin and/or anti-interleukin-1, to caecal-ligated and punctured rats may clarify the
specific action of both 'hormones' upon hepatic metabolism in vivo , and their role in the
modulation of the metabolic response.
Is the metabolic response to sepsis adaptive ?
The magnitude of the stress imposed by sepsis is a function of the size of the
infectious process, the number of invading organisms and their virulence. The severity of
infection modulates the metabolic response, for example increasing the resting energy
expenditure and the extent of the negative nitrogen balance (Elwyn, 1980). The effort to
contain and eliminate invading microorganisms is directly related to their number and
virulence, so that whereas a few thousand microorganisms will be contained by
phagocytosis, many million require mobilisation of both cellular and humoral immune
responses. Wiles et al (1980) hypothesized that the systemic response to sepsis is largely
independent of the type of invading organism. In favour of this view is the similarity in
many of the metabolic and physiological changes in gram-positive, gram-negative,
anaerobic, fungal and even viral sepsis (Deutschman et al , 1987), suggesting that the
series of responses to infection are primarily a function of the septic animal. Therefore, if
the metabolic response to sepsis is considered to be uniform, despite the nature of the
infective agent, it would appear to be adaptive and protective.
It is probable that an injured or infected animal in the wild, under conditions
where food is not immediately available, could starve. It seems to be adaptive, that in order
to maintain energy production and cell function throughout the body, protein, especially
from skeletal muscle, is catabolised. However, prolonged proteolysis can lead to an
impaired immunological capacity and to perpetuation of the infectious process (Chapter 1;
Kettlewell et al , 1979).
Ketone bodies are able to decrease skeletal muscle amino acid release, therefore
decreasing the availability of precursors for gluconeogenesis (Williamson et al , 1978).
The fact that ketogenesis is depressed by sepsis during the starved state (Chapter 5) might
also be regarded as detrimental. The administration of exogenous ketone bodies was,
however, unable to reduce proteolysis and gluconeogenesis in fasted-infected sheep
(Radcliffe et al , 1981 and 1983).
The progression of the septic process to the development of multiple organ failure
(Duff, 1985) and death, might be considered "maladaptive" (Wilmore et al , 1983), but
more likely represents the effects of overwhelming the adaptive responses to sepsis.
The attenuation of many of the classical postoperative metabolic responses, such
as increased cortisol release and negative nitrogen balance, does not seem to cause harmful
effects following elective operative procedures (Kehlet et al , 1980).
With modern intensive therapy, supported by high-technology medicine, the
possibility of manipulating the metabolic response to sepsis may become part of the
therapeutic strategy. The exaggerated and possibly harmful metabolic response could be
attenuated if the patient's temperature and substrate flow were adjusted by provision of
exogenous heat and substrates, the latter in the form of adequate
nutritional support (Wilmore et al , 1983). The development of non-invasive techniques,
such as bed-sided 'metabolic carts', make it possible to monitor energy substrates' kinetics
(Elwyn et al , 1981; Stein, 1986). Some of the possible sites for modifying the metabolic
response to sepsis are illustrated in Fig. 8.2.
The use of a number of biological response modifiers has been discussed in the
introductory Chapter, and considerable research has been devoted to assess their effect in
the metabolic response to injury/sepsis (Nohr & Meakins, 1985).
The administration of large doses of steroids in sepsis is common practice,
especially in septic shock (Forse & Kinney, 1985). Recent evidence supports, at least in
theory, the use of steroids because
of
their
actions,
such as
the
ability
to
induce key
gluconeogenic enzymes (Shackelford et al , 1986), to inhibit oxygen-free radical
generation (Karakusis, 1986), to block prostaglandin and leukotriene synthesis (HallaAngeras et al , 1986; Samuelsson, 1983),
Fig. 8.2.
and to block cachectin formation by macrophages (Cerami et al , 1986; Beutler & Cerami,
1987). In order to be able to block cachectin synthesis in vitro , however, macrophages
have to be exposed to the effects of steroids before their activation by the bacterial product
(LPS) (Fig. 8.1.). Support for the use of steroids at an early stage of infection, is the
finding that an early intravenous bolus of methylprednisolone administered at the
time of the operation, significantly improved the survival of caecal-ligated and punctured
rats, compared to infusing the drug continuously over the 7 day study period (Hollenbach
et al , 1986). In contrast to the experimental situation, however, patients almost always
present themselves with well established infection when many machrophages have already
been activated.
Cachectin has been proposed as an important mediator of the changes brought
about by infection (Beutler et al , 1986; Beutler & Cerami, 1987). It would seem therefore
conceivable that neutralizing monoclonal antibodies against human cachetin might prove
useful in the treatment of sepsis in its early stages (Fig. 8.2.).
In the present study many of the metabolic changes in response to sepsis were
reversed by the treatment to lower the plasma insulin (Chapter 7), either by
mannoheptulose or streptozotocin (Fig. 8.2.). Streptozotocin leads to permanent damage of
pancreatic ß-cells (Weiss, 1986), and its clinical use is, therefore, impossible. On the other
hand, mannoheptulose has been studied in man (Viktora et al , 1969; Johnson et al , 1969;
Johnson & Wolff, 1970; Lev-Ran et al , 1970). Whether its use in septic patients will cause
reversal of some of the metabolic changes in the liver is not known. Mannoheptulose
causes hyperglycaemia, which could prohibit its use in septic patients with established
glucose intolerance. One advantage in the possibility of manipulating the plasma insulin
concentrations
during sepsis, is the fact that, as opposed to the activation of macrophages and the
consequent release of interleukin-1 or cachectin,
which are early events in the establishment of the septic state, the increased plasma insulin
concentration, induced by sepsis, is present in the hyperdynamic septic state, falling only in
the hypometabolic late sepsis (Chaudry et al , 1979). Thus, insulin modulation could
theoretically be used as a way to modify the metabolic response in moderately and severely
septic patients (Fig. 8.1.). Nevertheless, any attempt to modify the metabolic response to
sepsis must consider the stage and severity of infection, the nutritional and hormonal status
of the patient, and which of the unwanted metabolic changes have been induced.
CHAPTER 8
General Discussion
The septic rat model and its metabolic implications
The experimental model used in the work presented in this Thesis
induced a moderate form of sepsis, as compared with that of others (Chaudry et al
, 1979; Wichterman et al , 1980). The septic rats did not become hypothermic nor
shocked. The blood lactate in the septic group was increased as early as 12 h after
the operation but never reached values defined as those for lactic acidosis (5
mmol/l). They did not go through the late hypodynamic septic state (Chaudry et al
, 1979), demonstrated by the fact that they did not develop hypoglycaemia or
hypoinsulinaemia at any time point studied.
The results demonstrate that gluconeogenesis and ketogenesis in rats
made septic by caecal ligation and puncture are impaired in vitro , whereas,
there are increased rates of lipogenesis and cholesterogenesis in vivo in the septic
rats. Therefore, although deprived of food, the septic rats, behaved more like fed
than fasted rats in terms of hepatic metabolism.
Increased
concentrations
of
blood
and
liver
metabolites involved in
gluconeogenesis - the new steady state
The catabolism induced by sepsis increased the availability of substrates,
such as pyruvate, lactate and alanine to the liver. The concentration of most of the
metabolites involved in the gluconeogenic pathway rose within the liver, but no
change was observed in either blood or hepatic glucose concentrations. The
pattern of accumulation of glucose precursors up to the level of glucose-6phosphate, without changes in hepatic glucose concentration, suggests a new
steady state has been achieved with higher concentrations of precursors.
Hepatocyte behavior during sepsis
The results clearly demonstrated that there is impairment in the hepatic
capacity for gluconeogenesis and ketogenesis in vitro in septic rats.
In the case of gluconeogenesis, the inhibition in vitro was progressive
with time, and affected all substrates tested (Chapter 4). There is evidence that
pneumoccocal infection in the rat decreases the hepatic activity of glucose 6phosphatase by 50% (Canonico et al , 1977), and that endotoxin, injected in mice,
decreases the hepatic activity of the gluconeogenic enzymes, namely, glucose 6phosphatase, fuctose 1,6-bisphosphatase and phosphenolpyruvate carboxykinase
(Shackleford et al , 1986). The hepatic concentration of acetyl-CoA, an important
activator of pyruvate carboxylase, is decreased at 48 h after the induction of
sepsis (Chapter 3). These alterations in key gluconeogenic enzymes may,
therefore, explain the decreased glucose synthesis in vitro .
The absence of hypoglycaemia in the septic rats suggests that this
impaired gluconeogenesis may be overcome in vivo , at least during moderate
sepsis, by increased concentrations of precursors, especially lactate and alanine
(Chapter 3), which are supplied in increased amounts from muscle (Beisel &
Wannemacher, 1980). The studies incubating isolated hepatocytes with lactate, at
concentrations similar to those found in the blood of the septic and sham-operated
rats in vivo , also supported the view that the depression in glucose synthesis can
be overcome in vivo (Chapter 4). Whether this effect of sepsis reducing the
hepatocyte ability to promote glucose synthesis may play a homeostatic role,
leading to the new steady state described (Chapter 3) and avoiding uncontrolled
hyperglycaemia, is still not clear.
However, several turnover studies in vivo have shown increased
gluconeogenic rates in sepsis, which conflicts with the findings in this study
(Wannemacher et al , 1980; Kelleher et al , 1982; Lang et al , 1984; Shaw et al ,
1985). The increase in glucose synthesis may be due to inappropriately low
insulin levels for the metabolic circunstances prevailing, for example the presence
of hyperglycaemia, in these turnover studies. Furthermore, some of these studies
report either mild or initial forms of sepsis, almost always associated with injury.
Therefore, as there is a lag period before the gluconeogenic enzymes become
inhibited (Canonico et al , 1977) leading to the depression in gluconeogenesis
(Chapter 4); substrate drive, at a time when gluconeogenic precursors are
available from peripheral catabolism, may become the predominant effect, as in
pure injury. During moderate forms of sepsis, as described in this study,
gluconeogenesis is impaired, but this can be overcome, so that glucose synthesis
can be maintained (Chapter 4). In severe forms of sepsis, the capacity to
overcome the gluconeogenic inhibition no longer exists; the compensatory
mechanisms, for example increased precursor availability, are overwhelmed
leading to profound hypoglycaemia and death. Therefore the severity of sepsis,
and the degree of stress induced by injury to which sepsis is often associated,
appear to be of fundamental importance in the modulation of the metabolic
response to sepsis. This view is supported by studies measuring rates of
gluconeogenesis in vivo from 14C-alanine (Wannemacher et al , 1980). In these
studies gluconeogenesis was increased in fasted rats which received a smaller
dose of Streptoccocus pneumonie , whereas glucose synthesis was decreased in
vivo when the dose of this microorganism was increased. The importance of the
severiy of infection is also illustrated by Wilmore's study (1976), in which nonshocked patients with burns and gram-negative sepsis exhibited decreased
gluconeogenesis in vivo leading to hypoglycaemia.
Sepsis also depressed ketogenesis in vitro (Chapter 5). Decreased ketone
body formation from long-chain fatty acids (oleate) in hepatocytes from septic
rats was observed 12 h after the operation, and the inhibition was not enhanced at
the later time points (Chapter 5). As this decrease in ketogenesis was not observed
with the short-chain fatty acid, butyrate, it is likely that entry of long-chain acylCoA into the mitochondria for oxidation is diminished during sepsis. Whether this
is induced by modulation of carnitine acyltransferase I activity, via increased
malonyl-CoA (McGarry & Foster, 1980; Vary et al , 1986) is still an open
question.
Changes in hepatic lipogenesis and cholesterogenesis in vivo
Sepsis stimulated the rates of hepatic lipogenesis and cholesterogenesis
in vivo . Increased hepatic lipogenesis in vitro has been reported in pneumoccocal
infection in the rat (Canonico et al , 1977). Sepsis has also been claimed to
increase the activities of acetyl-CoA carboxylase and lipid synthetase (Pace et al ,
1981 ), the former enzyme is subject to short-term activation by raised insulin
concentrations (Brownsley & Denton, 1987). In the present study, sepsis
increased the rate of synthesis of the non-saponifiable as well of the saponifiable
lipids (Chapter 6). The increased hepatic cholesterogenesis in vivo may reflect
the increased demand for cholesterol in the catabolic state of sepsis, for
membrane synthesis and the proliferation of cells in the reticuloendothelial
system. Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme
in cholesterogenesis, and its activity is increased by sepsis (Chapter 6; Canonico
et al , 1977). This increased HMG-CoA reductase activity appeared to be related
to total enzyme concentration, rather than changes in the enzyme activation state
(Chapter 6).
A unified hypothesis for the regulation of the changes in hepatic metabolism in
response to sepsis - the action of insulin.
The key factor which may prevent the septic rats responding in the same
way as the sham-operated rats to starvation is the raised plasma insulin. This
increase in plasma insulin is likely to be due to stimulation of pancreatic secretion
by bacterial products, such as LPS (Yelich & Filkins, 1980) and leucocyte
endogenous mediator, which contains interleukin 1 (George et al , 1977).
Insulin is known to stimulate lipogenesis and cholesterogenesis and
decrease gluconeogenesis and ketogenesis by its short-term actions on the
activation state of key enzymes, such as acetyl-CoA carboxylase, and by longerterm regulation of enzyme concentration. Although plasma glucagon is also
increased by sepsis (Beisel & Wannemacher, 1980), its stimulatory effects on
ketogenesis and gluconeogenesis and its inhibitory effects on lipogenesis may be
antagonised by the raised plasma insulin.
The results from septic rats treated with either mannoheptulose or
streptozotocin (Chapter 7), with the aim of decreasing the plasma insulin, support
the view that hyperinsulinaemia is a key factor in the metabolic changes in the
liver induced by sepsis.
Treatment to lower plasma insulin produced a decrease in hepatic
pyruvate concentration, and changes in the new steady state of gluconeogenic
intemediates in sepsis by activating the glucose 6-phosphate/glucose cycle,
leading to a decrease in glucose 6-phosphate concentration and the consequent
hyperglycaemia (Chapter 7) suggesting increased glucose synthesis. On the other
hand, Wannemacher et al (1980) measuring 14C-alanine incorporation into
glucose, in vivo , in rats pre-treated with mannoheptulose and injected with
Streptoccocus pneumoniae (104), showed no increase in 14C incorporation into
glucose, as compared to controls. However, rats receiving 107 Streptoccocus
pneumoniae had a significantly lower incorporation of 14C into glucose and
when compared to heat-treated controls. Therefore mannoheptulose treatment
caused no appreciable change in gluconeogenic rates in vivo with the milder
bacterial treatment, whereas with the more severe bacterial injection
gluconeogenic rates were decreased in mannoheptulose treated rats in vivo .
Mannoheptulose was therefore not able to help to overcome the decreased
gluconeogenic rates found in very severe sepsis.
Septic rats, deprived of food, and treated to lower plasma insulin levels
had blood and hepatic ketone body concentrations similar to those of shamoperated rats (Chapter 7). As the hepatic lipogenic rates were decreased by the
same treatment (Chapter 7), possibly decreasing malonyl-CoA concentration, it is
possible that
ketogenesis was reactivated in the septic treated rat, perhaps as a
result of more long-chain acyl-CoA entering the mitochondria for oxidation.
Lowering plasma insulin concentration also induced a reduction in the rates of
cholesterogenesis in vivo in the liver of septic rats (Chapter 7).
Is the metabolic response to sepsis adaptive ?
The magnitude of the stress imposed by sepsis is a function of the size of
the infectious process, the number of invading organisms and their virulence. The
severity of infection modulates the metabolic response, for example increasing
the resting energy expenditure and the extent of the negative nitrogen balance
(Elwyn, 1980). The effort to contain and eliminate invading microorganisms is
directly related to their number and virulence, so that whereas a few thousand
microorganisms will be contained by phagocytosis, many million
require
mobilisation of both cellular and humoral immune responses. Wiles et al (1980)
hypothesized that the systemic response to sepsis is largely independent of the
type of invading organism. In favour of this view is the similarity in many of the
metabolic and physiological changes in gram-positive, gram-negative, anaerobic,
fungal and even viral sepsis (Deutschman et al , 1987), suggesting the series of
responses after the septic insult are primarily a function of the septic animal.
Therefore, if the metabolic response to sepsis is considered to be uniform, despite
of the nature of the infective agent, it would appear to be adaptive and protective.
It is probable that an injured or infected animal in the wild, under
conditions where food is not immediately available, could starve. It seems to be
adaptive, that in order to maintain energy production and cell function throughout
the body, protein, especially from skeletal muscle, is catabolised. However,
prolonged proteolysis can lead to an impaired immunological capacity and to
perpetuation of the infectious process (Chapter 1; Kettlewell et al , 1979). As
sepsis sometimes may be associated with renal failure, the protein catabolism
induced by sepsis under these circunstances might be considered as maladaptive
(Wilmore et al , 1983), or more properly, that the adaptive mechanisms were
overwhelmed. Ketone bodies are able to decrease skeletal muscle amino acid
release, therefore decreasing the availability of precursors for gluconeogenesis
(Williamson et al , 1977). The fact that ketogenesis is depressed by sepsis during
the starved state (Chapter 5) might also be regarded as detrimental. The
administration of exogenous ketone bodies was, however, unable to depress
proteolysis and gluconeogenesis in fasted-infected sheep (Radcliffe et al , 1981
and 1983). The progression of the septic process to the development of multiple
organ failure (Duff, 1985) and death, might be considered a mal adaptation, but
more likely represents the effects of overwhelming the adaptive responses to
sepsis.
The attenuation of many of the classical postoperative metabolic
responses, such as increased cortisol release and negative nitrogen balance, does
not seem to cause harmful effects following elective operative procedures (Kehlet
et al, 1980).
With modern intensive therapy, supported by high-technology medicine,
the possibility of manipulating the metabolic response to sepsis may become part
of the therapeutic strategy. The exaggerated and possibly harmful metabolic
response could be attenuated if the patient's temperature and substrate flow were
adjusted by provision of exogenous heat and substrates, the latter in the form of
adequate
nutritional support (Wilmore et al , 1983). The development of non-invasive
techniques, such as bed-sided 'metabolic carts', make possible to monitor energy
substrates kinetics (Elwyn et al , 1981; Stein, 1986). Some of the possible sites for
modifying the metabolic response to sepsis are illustrated in Fig. 8.1.
Fig. 8.1.
The use of a number of biological response modifiers has been discussed in the
introductory Chapter, and intense research has been devoted to assess their effect
in the metabolic response to injury/sepsis (Nohr & Meakins, 1985).
The administration of large doses of steroids in sepsis is common
practice, especially in septic shock (Forse & Kinney, 1985). Recent evidence
supports, at least in theory, the use of steroids because
of
their
actions,
such as the ability to induce key
gluconeogenic enzymes (Shackelford, 1986), to inhibit oxygen-free radical
generation (Karakusis, 1986), to block prostaglandin and leukotriene synthesis
(Halla-Angeras et al , 1986; Samuelsson, 1983), and to block cachectin formation
by macrophages (Cerami et al , 1986). In order to be able to block cachectin
synthesis in vitro , however, macrophages have to be exposed to the effects of
steroids before their activation by the bacterial product (LPS) (Fig. 8.1.). Support
for the use of steroids at an early stage of infection, is the finding that an early
intravenous bolus of methylprednisolone administered at the time of the
operation, significantly improved the survival of caecal-ligated and punctured
rats, compared to infusing the drug continuously over the 7 day study period
(Hollenbach et al , 1986). In contrast to the experimental situation, however,
patients almost always present themselves with well established infection when
many machrophages have already been activated.
Cachectin has been proposed as an important mediator of the changes
brought about by infection (Cerami et al , 1986; Beutler & Cerami, 1987). It
would seem therefore conceivable that neutralizing monoclonal antibodies against
human cachetin might prove useful in the treatment of sepsis in its early stages
(Fig. 8.1.).
In the present study many of the metabolic changes in response to sepsis
were reversed by the treatment to lower the plasma insulin (Chapter 7), either by
mannoheptulose or streptozotocin (Fig. 8.1.). Streptozotocin leads to permanent
damage of pancreatic ß-cells (Weiss, 1986), and its clinical use is, therefore,
impossible. On the other hand, mannoheptulose has been studied in man (Viktora
et al , 1969; Johnson et al , 1969; Jonhson & Wolff, 1970; Lev-Ran et al , 1970).
Whether its use in septic patients will cause reversal of some of the metabolic
changes in the liver is not known. Mannoheptulose causes hyperglycaemia, which
could prohibit its use in septic patients with established glucose intolerance.
Diazoxide did not have any appreciable effect on the metabolic changes in the
septic rats (Chapter 7), perhaps because of its indirect hyperglycaemic effect from
stimulation of catecholamines, which in turn, inhibit insulin secretion (Henquin et
al , 1982). One advantage in the possibility of manipulating with the plasma
insulin concentrations during sepsis, is the fact that, as opposed to the activation
of macrophages and the consequent release of interleukin-1 or cachectin, which
are early events in the establishment of the septic state, the increased plasma
insulin concentration, induced by sepsis, is present along with the hyperdynamic
septic state, falling only in the hypometabolic late sepsis (Chaudry et al , 1979).
Thus, insulin modulation could theoretically be used as a way to modify the
metabolic response in moderately and severely septic patients (Fig. 8.1.).
Nevertheless, any attempt to modify the metabolic response to sepsis
must consider the stage and severity of infection, the nutritional and hormonal
status of the patient, and which of the unwanted metabolic changes have been
induced.
CHAPTER 9
Common features of the changes in hepatic metabolism induced by sepsis
and by total parenteral nutrition in the human
Introduction
Total Parenteral Nutrition (TPN) is an accepted treatment for patients with
malnutrition and intestinal failure (for review see: Grant & Todd, 1982; Silk,
1983; Phillips & Odgers, 1986). Controversy surrounds the choice of nutrients
and the rates of administration (Elwyn, 1980; Wolfe et al , 1980; Kettlewell,
1982). Recent evidence, from experimental work (including the results already
presented in this Thesis) and clinical studies, suggest some similarity in the
effects of sepsis on hepatic metabolism compared to those of the fed state
promoted by intravenous feeding (Fig. 9.1.).
Fig. 9.1.
Hepatic dysfunction, including increases in serum levels of liver
enzymes, has been consistently reported in experimental animals (Meurling &
Roos, 1981; Boelhouwer et al, 1983) and patients receiving Total Parenteral
Nutrition (Grant et al, 1977; Sitges-Serra et al , 1983; Robertson et al , 1986).
Hepatic dysfunction has also been reported during sepsis (Silen & Skillman,
1976; Royle & Kettlewell, 1980; Forse & Kinney, 1985). In addition,
parenterally fed patients with major sepsis were found to have almost double the
incidence of abnormal liver function tests compared with patients with no
evidence of sepsis, but also receiving intravenous feeding (Robertson et al ,
1986).
Sepsis increases substrate availability and elevates plasma insulin
(Chapter 3) concentrations which, in turn, stimulate hepatic lipogenesis in vivo
(Chapter 6). Esterification of the newly formed non-esterified fatty acids is also
increased by sepsis (Chapter 5), and perhaps, the accumulation of lipids found
during sepsis (Guckian et al , 1973; Campion , 1976) may well reflect an
imbalance in the liver between production and the capacity to export
triacylglycerol. Total Parenteral Nutrition also increases hepatic lipogenesis and
fat accumulation in the liver particularly when excess carbohydrate intake is
transformed into fat (Elwyn et al , 1981; Stein & Mullen, 1985). This becomes the
steatosis seen in liver biopsies (Benotti et al , 1976; Maini et al , 1976; Grant et al
, 1977). Hall et al (1984) have shown that the liver steatosis in rats, induced by
TPN, was due to enhanced hepatic synthesis of fatty acid and reduced
triacylglycerol secretion. Hepatic acetyl-CoA carboxylase activity increases in
parenterally nourished rats, and is positively correlated with hepatic lipid content
(Hall et al, 1984). It is also interesting that acetyl-CoA carboxylase and fatty acid
synthetase have increased activity in the fasted-infected rat (Pace et al , 1981). It
is important to emphasize that acetyl-CoA carboxylase is subject to short-term
regulation by insulin, the latter increasing its activity in the liver both in vivo and
in vitro (Brownsey & Denton, 1987)
Sepsis impairs ketogenesis in vitro (Chapter 4) and causes a decrease in
blood ketone bodies despite starvation (Chapter 3). Blood ketone bodies are also
low in patients fed parenterally, probably due to inhibition of lypolysis by
elevated plasma insulin concentrations (Maini et al , 1976).
Therefore, the factor bringing about these shared changes (Fig. 9.1.) is
probably the increased plasma insulin concentrations present during sepsis (Beisel
& Wannemacher, 1980; Neufeld et al , 1982; Shaw et al , 1985; Long et al , 1985;
Frayn et al , 1986) and in parenterally fed patients (Maini et al , 1976; Page &
Clibon, 1980). The hyperinsulinaemia of sepsis may be induced by stimulation of
the ß-cell by bacterial products (endotoxin, leucocytic endogenous mediator)
causing increased insulin secretion (George et al , 1977; Yelich & Filkins, 1980),
whereas, patients receiving intravenous feeding appear to increase their plasma
insulin concentrations in response to the considerable amount of glucose infused
continuously to meet the caloric needs (Sanderson & Deitel, 1974; Maini et al ,
1976).
It may be beneficial, therefore, to lower the elevated concentrations of
plasma insulin, by diminishing the exogenous-glucose stimulatory effect on ßcells, in patients fed parenterally. This was attempted in a clinical study either by
reducing glucose intake, by offering lipid as part of the caloric source, or by
promoting a period free of glucose infusion during intermittent or cyclical TPN.
Abnormalities of liver funtion developing in parenterally fed patients during
sepsis were also studied.
CYCLICAL PARENTERAL NUTRITION
"When we are forced to do something artificial we must aim
at remainning as close as possible to the natural situation."
Professor Sir Hans Krebs [1900-1981]
Continuous infusion of nutrients throughout 24 h imposes important
changes in the hormonal milieu comparable with a "steady fed state" (Page &
Clibon, 1980). Man is a meal eater and physiologically alternates periods of
feeding with post-absorptive periods.
Classical continous TPN may reasonably be criticised on the grounds it
does not allow for any post-absorptive period, and therefore maintains patients in
a store mode, inhibiting the state where nutrients are redistributed. This practice
evolved following the classical studies of Dudrick et al
(1969), who
demonstrated that growth and nitrogen balance could be achieved with the
continuous infusion of glucose and amino acids.
Recently, it is becoming common practice to provide intravenous nutrition
intermittently (Cyclical Parenteral Nutrition), to selected groups of hospital
patients, usually over 12-14 h (Maini et al , 1976; Page & Clibon, 1980;
Matuchanski et al , 1981 and 1985). In addition to allowing a period of freedom
from intravenous appendages, enhancing patients' mobility and morale, Cyclical
TPN also provides a 'post-absorptive' period which is theoretically closer to the
physiological meal pattern. The criteria for selection of hospital patients to
receive Cyclical Parenteral Nutrition (adapted from Page & Clibon, 1980) are
illustrated in Fig. 9.2.
Fig. 9.2.
This form of TPN administration was originally developed for home
patients requiring intravenous feeding. In 1973 Jeejeebhoy et al reported a
successful case of home parenteral nutrition, when cyclical intravenous feeding
was the method of administration. The introduction of a Dacron-cuffed, silicone
ruber catheter (Broviac, 1973) to deliver TPN solutions into a large vein for long
periods of time was an important step for home parenteral feeding. Today,
selected patients with intestinal failure can be successfully maintained in good
health and full activity (For review on Home Parenteral Nutrition see: Lees et al ,
1981; Johnston, 1982; Silk, 1983; Phillips & Odgers, 1986).
To date some 200 patients have been fed parenterally at home in the
United Kingdon and Ireland, between 1977 and 1986 (Mughal & Irving, I987),
with the main indications illustrated in Fig. 9.3. Despite being fairly widely used,
many of the metabolic effects of intermittent intravenous feeding, as well as,
some of the basic information, such as the optimal essential trace element
provision to home TPN patients (Shenkin et al, 1986), are not yet clear. Messing
et al (1983) have shown that in mildly catabolic patients, cyclical TPN promoted
a gain in visceral and lean body mass, similar to that obtained by continuous TPN.
Matuchansky et al (1981) also reported that nocturnal cyclical TPN was safe,
efficient and psychologically well tolerated in malnourished patients with
severe gastrointestinal disorders. Cyclical TPN has also been used successfully
in children undergoing bone marrow transplantation, when an infusion-free period
is required for the adminstration of drugs and blood transfusions without
interfering with the nutritional support (Reed et al , 1983).
Fig. 9.3.
Some of the possible metabolic differences between Cyclical Parenteral
Nutrition and Continuous TPN are illustrated in Fig. 9.4., as described by Page
and Clibon (1980).
Fig. 9.4.
Recour et al (1980) reported changes in blood and plasma metabolites in
two children on nocturnal cyclical TPN. They showed increases in insulin after
the start of the feeding period. During infusion, in contrast to fasting, there was an
increase in pyruvate and lactate, but decreases in free fatty acids and ketone
bodies. Benotti et al (1977) have shown improvement in hepatic function test
values during intermittent intravenous feeding, in patients fed initially
continuously followed by an identical period on cyclical TPN. In other studies,
glucose and hypertonic amino acid infusion was stopped for 8-10 hours during the
night, and blood and plasma metabolites were measured 3-4 hours after the
suspension of the infusion. There was a decrease in glucose and an increase in
insulin, but no change in fatty acids, alanine, lactate or ß-hydroxybutyrate levels
(Maini et al , 1976). However, during the glucose-free period, patients received
either 3% amino acids or oral protein (40-90 g / day). These investigators
suggested that during the 'post-absorptive', or infusion-free period, stored energy
such as fat and glycogen can meet the energy requirements and the infused
amino acids can be incorporated and preferentially used for visceral protein
synthesis, which suggests that Cyclical TPN is best for preserving visceral
protein. The mobilization of nutrients during the post-absorptive period of
intermittent intravenous feeding has been studied in children, using indirect
calorimetry. Glycogen was used and lipid stores mobilised during the post
absorptive period (Putet et al , 1984).
It is interesting that, during cyclical TPN, patients may show marked
losses of calcium in the urine and develop negative calcium balance as compared
to patients on continous TPN. What implications this might have on the metabolic
bone disease described on long-term home TPN patients requires further
investigation (Wood et al , 1985).
Essential fatty acid defficiency is a well known complication in patients
receiving lipid-free TPN regimens (Wene et al , 1975; Riella et al , 1975;
Gooodgame et al , 1978), but it may be corrected by either the inclusion of lipid
in the TPN regimen (Elwyn, 1980) or, alternatively, by the cutaneous application
of sunflower seed oil (Press et al , 1974). Mascioli et al (1979) have shown that
cyclical TPN, possibly due to the periodical reduction of insulin levels stimulating
fat mobilization, may be effective in preventing and treating fatty acid
defficiency.
Lanza-Jacoby et al (1985) demonstrated changes in the circadian
rhythmicity of liver weight, and protein and glycogen content in rats fed with
cyclical TPN. These rhythm patterns were similar in rats receiving cyclical TPN
as compared to those of rats fed with intermittent enteral feeding. Matuchansky et
al (1985) studied the effects of cyclical nocturnal TPN on circadian rhythms of
blood lipids, lipoproteins and alipoproteins in six patients who were receiving
cyclical intravenous feeding for at least a month. Although these rhythms may
represent endogenous bioperiodic phenomena, nocturnal cyclical TPN was
associated with the persistence of the circadian rhythms detected in control
hospital
patients
fed
3
meals
a
day.
Despite the increased interest in Cyclical TPN, it does not appear to be
clear whether the changes in blood metabolites and plasma insulin, during the 12
hour infusion-free period, can enhance the development of a 'post-absorptive'
period sufficient to produce the expected metabolic changes (Fig. 9.3.), to confirm
Cyclical TPN as the more physiological form of intravenous feeding
administration.
Experimental Design
To assess the contribution of sepsis to liver dysfunction during Total
Parenteral Nutrition, a large number of parenterally fed patients with sepsis was
studied. In addition, in an attempt to lower plasma insulin, and perhaps avoid
some of the unwanted effects of Total Parenteral Nutrition on hepatic metabolism,
two studies were designed. Each study was approved by the Ethics Committee of
the John Radcliffe Hospital, Oxford, and an informed consent was obtained from
each patient.
STUDY ON LIVER DYSFUNCTION DEVELOPING IN PARENTERALLY
FED PATIENTS DURING SEPSIS
Although abnormalities of liver function developing in patients on TPN
are common (Roberson et al , 1986), the time-course of the effect of sepsis on
liver function tests in TPN patients requires clarification. Therefore, in order to
evaluate how common altered liver function test values are, and how they
progress during continuous Total Parenteral Nutrition and during sepsis, a survey
was conducted of liver function tests in parenterally fed patients who were or
were not septic during their TPN course.
We have studied the serum albumin, bilirubin, aspartate transaminase
(AST), and alkaline phosphatase concentrations in adults receiving TPN. The
data were collected prospectively and the results analysed in two groups: nonseptic versus septic patients.
Patients
285 patients referred to the Oxford TPN Team in the period between 1978
and 1986 were studied.
Septic patients
Included in the septic group were 126 patients with clinical evidence of
sepsis, such as the discharge of pus from wounds or drainage tubes, or a pyrexia
greater than 37.5°C with positive bacterial cultures from pus and blood. Many of
the patients developed sepsis as a post-operative complication.
Non-septic patients
159 patients without any clinical evidence of sepsis were compared to the
septic group.
Parenteral Nutrition Regimen
Approximately 70% of the patients received the standard Oxford fat-based
TPN regimen (Harper et al , 1983). The other patients either received glucose as
their sole calorie source, or special regimens to meet their clinical situation, for
example patients in renal failure (Grant & Todd, 1982).
Measurements of liver function tests (LFTs)
Liver function tests were measured three times a week during the first
week on TPN, and twice a week thereafter. The mean of the results of LFTs
measured during weeks 1,2,3&4 of intravenous feeding were analysed in the
study.
RANDOMIZED
CONTROLLED
STUDY
ON
STANDARD
VERSUS
TAILORED TOTAL PARENTERAL NUTRITION
In this study the non-protein calorie intake was given either entirely as
glucose, or glucose and fat. An "individualized" or a "standard" regimen was
provided to those who had glucose as their sole calorie source. The results
obtained from these patients were compared to those obtained from patients who
received an equicaloric "standard" lipid TPN regimen containing fat, as half of the
non-protein calorie source.
Criteria for selection of patients and informed consent
Patients refered to the Oxford TPN team, requiring intravenous feeding
were randomly allocated to receive a standard glucose-based regimen, or a
tailored glucose-based regimen, or the standard Oxford lipid-based TPN regimen
(Harper et al , 1983). Patients with renal or liver impairment, patients with
diabetes mellitus, or critically ill patients were excluded from the trial. All
patients agreed to take part in the study after being informed on all the procedures
involved, as well as, receiving a detailed explanatory note.
Patients
Fifty patients were studied. Table 9.1. & 9.2. give the details about their
age, sex and TPN regimen received, and the main indications for TPN.
Table 9.1. Patients fed on tailored or standard TPN regimens
____________________________________________________________
Nº of Patients
Age
Sex (M/F) TPN Regimen
_____________________________________________________________
8
11
59.0±5.1
46.9±7.3
4:4
7:4
31
51.3±6.4
12:19
Tailored Glucose Regimen
Standard Glucose Regimen
Standard Lipid Regimen
Total = 50
_____________________________________________________________
Values for age expressed as Mean±S.E.M.
_____________________________________________________________
Main indications for TPN
_____________________________________________________________
Table 9.2.
Nº of Patients
___________________________________________
Standard Glucose
Tailored Glucose
Standard Fat
_____________________________________________________________
Inflammatory bowel disease
4
4
9
Post-operative complications
2
1
6
Injury
1
0
5
2
6
Other conditions
10
_____________________________________________________________________
__
Total Parenteral Nutrition Regimens
1. The Tailored Glucose Regimen was defined based on the estimated
resting energy expenditure, calculated using the Harris-Benedict (1919) equation
which takes into account the following variables: age, height, weight and sex
(Table 9.3.).
Table 9.3.
The Harris-Benedict Equation
__________________________________________________________
Daily REE for males = 66.4230 +013.7516 W + 5.0033 H - 6.7750 A
Daily REE for females = 655.0955 + 9.5634 W + 1.8496 H - 4.6756 A
__________________________________________________________
Where W = weight in Kg, H = height in cm, and A = age in years
Although differences are found between the the "calculated" values and those
obtained with indirect calorimetry using a "metabolic cart", the differences are not
large enough to invalidate the equation, especially when the estimated values are
corrected acording to the patients' clinical conditions (Elwyn, 1980; Stein, 1985).
Therefore, patients received 1.10 (post-operative patients) or 1.25 (depleted
patients) of their calculated Resting Energy Expenditure. The protein intake also
was adjusted to either 160 or 250 mg/kg body wt./day.
2. The Standard Glucose Regimen consisted of:
Nitrogen
14 g
Glucose
500 g
Sodium
100 mmol
Potassium
Chloride
100 mmol
124 mmol
Calcium
14 mmol
Phosphate
30 mmol
Zinc
Magnesium
0.2 mmol
19 mmol
Folic acid
15 mg
Fat emulsion 20%
100 g
Vitamin and mineral supplements were given as described by Harper et al
(1983). This standard glucose-based regimen supplied 4.2 MJ / 24 h (2000kcal /
24 h) of glucose (500 g). The glucose-amino
acid solution was made up in sterile conditions under a laminar flow hood to
volumes from 1.5 to 3 litres in a three litre bag. In order to avoid the development
of essential fatty acid deficiency both standard and tailored glucose-based
regimens included the infusion of one bottle of a 10% lipid emulsion, once a
week, starting at the end of the first week of feeding.
3. The Standard Lipid Regimen (Oxford Regimen) differed from the Standard
Glucose Regimen only in the non-nitrogen calorie source. Half the calories were
provided as a 20% lipid emulsion (Fat - 100 g). This lipid-based regimen has
been proved to be efficient in approximately 70% of patients who received TPN
in Oxford over a three year period (Harper et al , 1983).
Blood sampling and measurements
Blood samples were taken at about 10 a.m. three times during the first
week of feeding (Days 1,3,6), and twice a week thereafter (Days 10,14,18,etc.).
Betwen 1 and 2 ml of blood were immediately deproteinized at the bedside in a
previously weighed tube containing 5 ml of ice-cold perchloric acid (10%
weight/volume), and used for metabolite determination. Pyruvate, acetoacetate, ßhydroxybutyrate, lactate, alanine and glucose were assayed using standard
enzymatic methods in patients receiving both glucose-based regimens (For details
see Chapter 2). Ketone bodies were measured in patients receiving the lipidbased regimen. Other measurements were those required for normal clinical
management of TPN patients (Harper et al , 1983). Daily 24 h urine collection
was performed to measure urinary urea, and total urinary nitrogen loss
estimated (Lee & Hartley, 1975) using the formula:
___________________________________________________________
Urine urea (mmol/l) X 24 h urine volume (litres) X 0.028 X 6/5 = nitrogen (grams/24h)
___________________________________________________________
Blood samples were also analysed to determine liver function tests,
including albumin, bilirubin, aspartate transaminase and alkaline phosphatase.
CONTROLLED STUDY ON CONTINUOUS VERSUS CYCLICAL TPN
In this study TPN was given initially continuously, followed by a period
in which the nutritents were infused during the day only, and finally TPN was
again offered continuously (Fig. 9.5). Changes in blood metabolites and
plasma insulin were measured 3 hourly over 24 h. The aim of this study was
therefore to assess how physiological Cyclical TPN is, in providing a 'postabsorptive' period and lowering plasma insulin during the infusion-free period, to
study whether this might resolve some of the unwanted metabolic changes
induced by TPN.
Fig. 9.5.
Criteria for selection of patients and informed consent:
Patients who had received continuous TPN for not less than two weeks
and required at least a further 7 days more TPN, who were clinically and
metabolically stable, were included in the study. All patients agreed to take part in
the study after being informed on all the procedures involved, as well as,
receiving a detailed explanatory note.
Patients
Six patients were studied. Table 9.4. shows details about their age, sex and
underlying condition.
Table 9.4.
Patients who received cyclical parenteral nutrition
_____________________________________________________________
Patient
Age
Sex
Diagnosis
_____________________________________________________________
J.A.
64
F
Ca of pancreas - Whipple
operation
G.S.
45
F
Ca of oesophagus -
oesophagogastrectomy
B.W.
21
M
R.T.A. - Multiple fractures
G.S.
39
M
Superior mesenteric artery
thrombosis - Short gut
V.G.
75
F
Duodenal perforation +
intestinal obstruction
P.W.
44
W
Crohn's
Mean±S.E.M. 48±7.7
M/F=1
_____________________________________________________________
The study period was 7 days. On day one, patients remained on continuous TPN.
On days 2-6 TPN was given during the day only (9A.M. to 9 P.M.), and on day 7
patients were fed again continuously.
Blood sampling
Blood was sampled 3-hourly on days 1,2,6,7 (total of 32 ml a day) and
twice-a-day on days 3,4,5 (total of 8 ml a day) (Fig. 9.4.). The sequential blood
samples every 3 hours over 48 h were taken via a cannula (Venflon G 18 - Viggo)
inserted into a forearm vein under local anaesthesia, using a two syringe method.
The cannula was securely taped and attached via an extension tubing to a 10 ml
syringe with 5 ml of 0.9% sterile sodium chloride containing heparin (Hepsal 10units/ml). The dead space of the plastic cannula and the extension tubing was
1.2ml. Two ml of the dilute blood were therefore withdrawn and discarded before
each blood sample was taken with a new syringe. The cannula system was flushed
with Hepsal following each sampling to prevent blood clotting in the cannula or
tubing, and an end-tap fitted to the extension tubing, which was therefore locked,
bent into a loop, and taped to the forearm. Between 1 and 2 ml of blood were
immediately deproteinized at the bedside in a previously weighed tube containing
5 ml of ice-cold perchloric acid (10% weight / volume), and used for metabolite
determination. The remaining blood, from the 4 ml withdrawn in each sample,
was immediately centrifuged at 5°C at 3000 rpm for 10 min to separate the
plasma, which was then transferred to a new tube and stored at -20°C for the
insulin determination.
TPN Regimen
All patients received the same sandard TPN Oxford regimen (Harper et al
, 1983) already described.
Measurements
Measurements were those required for normal clinical management of
TPN patients (Harper et al , 1983). Daily 24 h urine collection was performed to
measure urinary urea, and total urinary nitrogen loss estimated using the Lee &
Hartley (1975) equation, already described. Twice weekly blood samples were
analysed to determine liver function tests, including albumin, bilirubin, AST and
alkaline phosphatase.
The deproteinized blood samples were used to determine the
concentrations of pyruvate, acetoacetate, ß-hydroxybutyrate, lactate, glutamate,
alanine and glucose using standard enzymatic methods (For details see Chapter
2). Plasma immunoreactive insulin was determined by a single anti-body
technique using charcoal binding (Albano et al , 1972 - For details see Chapter
2).
STATISTICAL ANALYSIS
Results were expressed as the mean ± the standard error of the mean
(S.E.M.) accompanied by the number of observations in parenthesis. The
representation used for mean values, and the mathematical definition of S.E.M.
are shown below:
Results were analysed using the Mann-Whitney U Test.
Results
The effect of sepsis on liver function tests in patients receiving total
parenteral nutrition
A survey was undertanken of ill patients receiving TPN in order to assess
the incidence of liver dysfunction induced not only by TPN but also the
contribution from sepsis.
The percentage of parenterally fed patients, who were septic or not, with
altered serum albumin concentration is shown in Figs. 9.6.
Fig. 9.6.
Patients with abnormal albumin during sepsis and TPN
80
*
Abnormal albumin (%)
60
²
²
Non-Septic
Septic
40
* P < 0.01
versus non-septic
20
² P < 0.01
versus week 1
0
1
2
3
4
Time of intravenous feeding (week)
The administration of Total Parenteral Nutrition had a beneficial effect in nonseptic patients by decreasing, with time, the percentage of patients with abnormal
(low) serum albumin concentration. More septic patients, however, remained with
abnormal albumin levels, perhaps, reflecting a higher degree of malnutrition in
these patients (Young & Hill, 1978), or the fact that severe malnutrition can be
perpertuated by sepsis (see Chapter 1), despite nutritional support.
The percentage of patients with abnormal serum bilirubin in shown in
Figs. 9.7.
Fig. 9.7.
Patients with abnormal bilirubin during sepsis and TPN
50
Abnormal bilirubin (%)
40
*
30
Non-septic
Septic
20
P < 0.01
versus non-septic
10
0
1 1
Week
2 2
Week
3 3
Week
4 4
Week
The percentage of non-septic patients with abnormal serum bilirubin increased
with time. More septic patients than non-septic patients presented abnormal
bilirubin during the first week of TPN.
The percentage of septic patients on parenteral nutrition who presented
with abnormal serum alkaline concentrations is illustrated on Fig. 9.8.
Fig. 9.8.
Patients with abnormal alkaline phosphatase during sepsis and TPN
100
²
Abnormal alk. phosph. (%)
80
²
*
Non-septic
Septic
60
*
40
* P < 0.01
versus non-septic
² P < 0.01
versus week 1
20
0
1 1
Week
2 2
Week
3 3
Week
4 4
Week
The proportion of both non-septic and septic parenterally fed patients with
abnormal alkaline phosphatase (elevated) levels, increased with time during the
first two weeks of feeding. More septic patients had raised alkaline phosphatase
than non-septic patients. However, as more non-septic patients developed
abnormal alkaline phosphatase levels with time, the percentage of patients with
this abnormality converged at the fourth and fifth week. This suggests sepsis
appears to promote the changes in alkaline phosphate at an earlier stage than TPN
alone.
The changes in the proportion of parenterally fed patients with serum
aspartate transaminase alterations, in the presence or absence of sepsis, is shown
in Fig. 9.9.
Fig. 9.9.
The proportion of septic patients receiving TPN with altered serum asparate
transaminase was higher than that of non-septic patients only in the second week
of intravenous feeding.
Tailored Glucose versus Standard Glucose TPN Regimen
Patients who received the tailored glucose regimen required slightly
longer period of feeding (21.5±6.21 days) than those who either received the
standard glucose TPN (17±2.42 days) or standard fat TPN (16.4±4.1 days Mean±S.E.M.). The nitrogen and caloric intake are presented in Table 9.5 .
Nitrogen and energy intake
___________________________________________________________
Table 9.5.
___
Nitrogen(g / d)
Glucose(g /d)
Kcal (d)
Kcal / N
___________________________________________________________
___
Glucose Tailored TPN *
12.28±1.10
375.2±26.21 1506.4±64.7
126.3±11.1
Glucose Standard TPN
14
500
2000
Lipid Standard TPN **
14
250
2000
142
142
______________________________________________________________________
___
* Results expressed as Mean value±S.E.M. ; ** In order to provide 2000 kcal/day , the
other half the non-nitrogen calories (1000 kcal/day) were offered as fat.
______________________________________________________________________
_
Table 9.6. gives the same data according to the body weight at the start of the
TPN course.
Nitrogen and energy intake according to the body weight
______________________________________________________________
Table 9.6.
Nitrogen (g/kg/d)
Glucose(g/kg/d)
Energy (kcal/kg/d)
______________________________________________________________
Glucose Tailored TPN
0.232±0.017
6.84±0.411
27.3±1.65
Glucose Standard TPN
0.424±0.165
8.93±0.568 *
37.7±2.27 *
______________________________________________________________________
___
* P < 0.05 ; compared to glucose tailored TPN
Patients who received the individualized glucose TPN regimen therefore received
significantly less glucose and calories than those who were on the standard
glucose regimen (Table 9.6).
Some of the patients receiving glucose as their sole non-nitrogen caloric
source required exogenous insulin at some stage of their feeding (Tailored
Glucose TPN - 3 / 8 (37.5%); Standard Glucose TPN - 3 / 11 (27.2%). This
compares unfavourably with only 9% in patients fed a standard lipid regimen
requiring insulin supplementation (Harper et al , 1983).
Providing tailored TPN did not cause appreciable change in the
concentration of blood glucose, lactate, pyruvate, alanine and ketone bodies (Figs
9.10., 9.11. and 9.12.). There was no appreciable day to day difference in the
results for each day. Therefore, data are presented as the mean for each day
studied (Day 1+3+6+10+14). Results from patients receiving exogenous insulin
were excluded.
Fig. 9.10.
Standard versus Tailored TPN- Blood Metabolites
6
µmol/ml whole blood
5
(24)
(16)
4
Standard TPN
Glucose
Tailored TPN
Glucose
3
2
(16)
(23)
1
0
Glucose
Lactate
X + S.E.M.
Blood alanine concentrations in patients receiving an individualized
regimen were slightly higher than those of patients receiving the standard glucose
regimen, perhaps reflecting the lower energy / nitrogen ratio in tailored glucose
patients (Table 9.5.).
Fig. 9.11.
Standard versus Tailored TPN - Blood Metabolites
0.5
(16)
0.4
(24)
0.3
Standard TPN
Glucose
Tailored TPN
Glucose
0.2
(23)
(16)
0.1
X + S.E.M.
0.0
Alanine
Pyruvate
Ketone body concentrations were also compared to those in patients
receiving the standard lipid regimen (Fig.9.12.)
Fig. 9. 12.
Standard versus Tailored TPN- Blood ketone bodies
(98)
0.10
0.08
0.06
(16)
Standard GGlucose
Tailored GGlucose
Standard Fat
(23)
0.04
X + S.E.M.
0.02
0.00
Ketone bodies
Providing half the non-nitrogen calories as fat increased ketone body
concentration, however, the difference did not reach statistical significance. The
fact that the ketone body levels in the lipid fed patients remained low was
probably due to the high carbohydrate load increasing plasma insulin, which in
turn, inhibited lipolysis and reduced the availability of fatty acids , the ketone
body precursors for the liver.
Standard versus Tailored TPN - Effect on Nitrogen Balance
Varying the non-protein caloric intake or individualizing the TPN regimen
did not produce any significant change in nitrogen balance, which was positive
overall (Fig. 9.13.). Patients receiving the higher carbohydrate intake had slightly
higher nitrogen retention.
Fig. 9.13.
Tailored versus Standard TPN - Effect on Nitrogen Balance
6
Nitrogen retained (g/day)
5
(34)
Standard Glucose
G
Taylored
Tailored glucose
Standard
Standard Lipid
Fat
(45)
4
(91)
3
X + S.E.M.
2
1
0
Nitrogen Balanc
Continuous versus Cyclical Parenteral Nutrition - Changes in blood
metabolites and plasma insulin over 24 h
Delivering TPN over 12 h during the day only (Cyclical TPN) produced
small changes in blood glucose concentration (Fig. 9.14.) which were not
statistically significant.
Fig. 9.14.
None of the patients had glycosuria and, therefore, did not require exogenous
insulin. Blood glucose concentrations were variable during the day and reached a
steady plateau during the night on continuous feeding.
Blood glucose
concentrations fell during the infusion-free period of Cyclical TPN, but this fall
was not statistically significant. Similar results have been reported by Byrne et al
(1981).
During Cyclical Parenteral Nutrition, blood concentrations of pyruvate
were similar to those during Continuous TPN. The provision of a 'post-absorptive
period' had no detectable effect on venous pyruvate concentration (Fig. 9.15.).
Fig. 9.15.
Increases in blood pyruvate concentration during the infusion period of two
children receiving Cyclical TPN have been reported by Recour et al (1980).
Blood glutamate was measured but no change in concentration was
observed during Cyclical TPN as compared to Continuous TPN (Fig. 9.16.)
Fig. 9.16.
Blood lactate concentration changes over 24 h of Cyclical versus
Continuous TPN are illustrated in Fig. 9.17. Lactate concentrations were slightly
higher during Continuous TPN compared to those during the infusion-free period
(Cyclical TPN OFF) but again the differences were not statisticaly significantly
significant.
Fig. 9.17.
Maini et al (1976) found no change in blood lactate during a 8-10 hour glucosefree period, whereas Recour et al (1980) reported increased blood lactate
concentration during the the infusion period in two children receiving Cyclical
TPN.
Changes in alanine concentration during 24 h are shown in Fig. 9.18.
Fig. 9.18.
Blood alanine levels were generally slightly higher during the infusion period and
fell during the post-absorptive period of Cyclical TPN, but this fall was not
statistically significant. Alanine concentration remained stable during Continuous
TPN.
The changes in plasma insulin concentration, over 24 h, induced by
offering TPN only during the day are shown on Fig. 9.19.
Fig. 9.19.
Plasma insulin concentration was fairly uniform during Continuous TPN. During
the feeding period of Cyclical TPN, when the infusion of nutrients doubles,
insulin concentration increased. During the 'post-absorptive' period insulin
concentrations decreased to concentrations as low as
those found after an
overnight fast (5 mU/l plasma - Record et al , 1973).
Ketone bodies remained very low at both Continuous and Cyclical TPN
(Fig. 9.20.). The decrease in insulin levels during the infusion-free period of
Cyclical TPN had no effect on blood ketone body concentration. However, during
the feeding period of Cyclical TPN, when the the rate of infusion is doubled as
compared to that of Continuous TPN, ketone body concentrations were slightly
lower than those found during Continuous TPN, although the differences were not
statistically significant.
Fig. 9.20.
Small numbers and marked individual variability in the metabolic
response tends to mask the overall effects of cyclical TPN. Therefore, all the data
from the "infusion" or "infusion free" period were aggregated and compared with
the aggregated data for continuous TPN. Pyruvate and glutamate concentrations
did not change (Figs. 9.21. & 9.22).
Fig. 9.21.
Fig. 9.22.
Lactate concentrations, however, were significantly decreased by
promoting a 'post-absorptive' period during Cyclical TPN (Fig. 9.23.).
Fig.9.23.
Blood alanine concentration increased when the rate of infusion was
doubled, during the feeding period of Cyclical TPN. The 'post-absorptive' period
decreased alanine concentrations (Fig. 9.24.). Similar increases in amino acid
concentration, by a factor of 1.5 with refeeding, have been reported (Newshome
& Leech, 1983).
Fig. 9.24.
Blood glucose concentrations remained unchanged during the feeding
period of Cyclical TPN, however, they decreased significantly during the 'postabsorptive' period, but not sufficiently to induce hypoglycaemia (Fig. 9.25.).
Fig. 9.25.
Plasma insulin concentrations increased significantly during the 'feeding
period' of Cyclical TPN, compared to those during Continuous TPN. On the other
hand they decreased dramatically in the infusion-free period (Fig. 9.26.).
Fig. 9.26.
Similar responses of insulin concentration to continuous TPN infusion (Dudrick
et al , 1970; Sanderson & Dietel, 1974; Byrne et al , 1981) and to intermittent
intravenous feeding (Maini et al , 1976; Byrne et al , 1981) have been reported.
The decrease in plasma insulin during the 'post-absorptive' period of
Cyclical TPN did not produce any appreciable change in the very low
concentrations of blood ketone bodies (Fig. 9.27.)
Fig. 9.27.
Urea excretion and nitrogen balance remained unchanged by Cyclical
TPN compared to Continuous TPN (Fig. 9.28.).
Fig. 9.28.
Continuous versus Cyclical TPN - Effect on Nitrogen Balance
2.4
(16)
Nitrogen retained (g/day)
2.0
(13)
Continuous TPN
Cyclical TPN
1.6
1.2
0.8
X + S.E.M.
( ) Nº of observation
0.4
0.0
Nitrogen
Changes in liver function during standard versus tailored Total Parenteral
Nutrition
Decreasing the carbohydrate intake by tailoring TPN did not produce an
appreciable effect on the concentrations of albumin or bilirubin (Fig. 9.29. &
9.30.).
Fig.9.29.
Fig. 9.30.
The bilirubin concentrations increased with time, and, in patients receiving the
standard lipid regimen were always slightly higher than those of patients on the
glucose-based regimens, but these differences
never reached statistical
significance. Tailoring TPN also did not alter the concentrations of alkaline
phosphatase and aspartate transaminase (Figs. 9.31. & 9.32.).
Fig. 9.31.
Fig. 9.32.
The same data are presented in a composite form in Table 9.7.
Table 9.7.
Tailored versus Standard TPN - Effects on liver function tests
___________________________________________________________
Results expressed as Mean±S.E.M. with number of observations in
parenthesis.
___________________________________________________________
Glucose Tailored
Glucose Standard Lipid Standard
______________________________________________________________________
Albumin (g / l)
31.1±0.52 (70)
31.0±1.0 (49)
29.5±0.61 (92)
Bilirubin (µmol / ml)
11.8±1.1 (64)
8.8±0.70 (49)
19.7±3.0 (90)
Alkaline phosphatase (IU / l)
300±25.5 (63)
377±37.3 (50)
228±11.1 (87)
Aspartate transaminase (IU / l) 27.9±2.1 (58)
49.9±8.8 (50)
46.3±8.5 (87)
______________________________________________________________________
Changes in liver function during continuous versus cyclical Total Parenteral
Nutrition
Liver function test values obtained from patients whilst on Continuous
TPN were compared to those when they were on Cyclical TPN. No statistically
significant changes were observed (Table 9.8.).
Table 9.8. Continuous versus Cyclical TPN - Effects on liver function
___________________________________________________________
Results expressed as Mean±S.E.M. with number of observations in
parenthesis.
___________________________________________________________
Continuous TPN Cyclical TPN
___________________________________________________________
Albumin (g / l )
32.6±1.9 (15)
36.2±1.3 (8)
Bilirubin (µmol / ml)
17.2±2.4 (21)
9.6±1.3 (10)
Alkaline phosphatase (IU /l)
411±38.6 (23)
316±51.7 (9)
Aspartate transaminase (IU / l) 28.9±3.9 (20)
40.0±10.1 (9)
___________________________________________________________
_
Discussion
Most patients can handle fairly large quantities of glucose, fat and amino
acids infused intravenously, and retain nitrogen, maintain or gain weight and
avoid glycosuria (Harper et al , 1983). In the present study, patients receiving
glucose or glucose and fat as their source of non-nitrogen calories, also retained
nitrogen and demonstrated a positive nitrogen balance (Fig. 9.13.).
Nevertheless, parenteral nutrition is invasive and unphysiological and not
surprisingly, therefore, the literature abounds with the problems caused by this
form of nutrition (Grant & Todd, 1982; Silk, 1983; Philips & Odgers, 1986).
Perhaps hepatic dysfunction with altered biochemical liver function tests (LFTs)
is the commonest recorded metabolic complication of intravenous nutrition
(Robertson et al ,1986). There is also a common association between sepsis and
abnormal liver function tests (Royle & Kettlewell, 1980).
In the present survey, the proportion of septic patients with low albumin
concentrations at the start of their parenteral nutrition course was similar to that of
non-septic patients, but significantly more septic patients had abnormal bilirubin
concentrations over the first week of intravenous feeding (Figs. 9.6.& 9.7). TPN
caused a progressive increase, with time, in the proportion of patients with
alkaline phosphatase abnormalities in both septic and non-septic patients.
However a significantly higher proportion of septic patients had raised alkaline
phosphatase levels in the early stages of TPN, when compared to the non-septic
patients (Fig. 9.9.).
Modulating the carbohydrate intake of parenterally fed patients did not
prevent the alterations in hepatic liver function tests (Table 9.6.), such as
progressive rises in liver enzymes (Harper et al , 1983; Robertson et al , 1986),
indicating that there was no advantage in decreasing the glucose load
(approximately 25% in patients receiving individualized glucose prescriptions Tables 9.5. & 9.6.; and 50% in patients receiving half their non-protein calories as
fat) in this respect. Offering a 'post-absorptive' period during Cyclical TPN also
did not alter liver function tests (Table 9.8.), compared to those measured during
Continuous TPN. Possibly the period of Cyclical TPN was too short (5 days) to
promote such changes. Benotti et al (1976) claim to have improved liver function
tests of patients who were receiving continuous TPN for more than 3 weeks, and
were converted to cyclical TPN for a further 3 weeks. Against this view is the fact
that alterations in liver function tests are also reported in home TPN patients
(Müller et al , 1982). Furthermore, other factors have been implicated in these
hepatic alterations, including nitrogen sources, the lack of any oral intake,
intestinal overgrowth by bacteria, sepsis, and additives used in the preparation of
some intravenous regimens (Robertson et al , 1986).
Decreasing the carbohydrate load, by tailoring the TPN regimen, caused
no changes in blood metabolites, particularly glucose, lactate, pyruvate, alanine,
and ketone bodies. This absence of a rise in ketone body concentrations may be
due to inhibition of lypolysis by the hyperinsulinaemia induced by the
carbohydrate load given to tailored TPN patients. Urea excretion and nitrogen
balance were not significantly different with any of the forms of TPN studied
(Fig. 9.13. & 9.28.).
No tapering off period was used when stopping the infusion in Cyclical
TPN patients for this study. One episode of rebound hypoglycaemia was
observed, which has been described by others (Dudrick et al , 1970; Sanderson &
Dietel, 1974). However, in a recent study involving 48 patients, Wagman et al
(1986) reached the conclusion that acute discontinuation of TPN infusion was a
fairly safe procedure.
The faster infusion of nutrients during cyclical TPN (12 hours - feeding
period) did not induce hyperglycaemia because plasma insulin levels rose "pari
passu", which in turn, illustrates a normal insulin secretory response to the
glucose load in these patients. This elevation in plasma insulin during the 12hinfusion period of Cyclical TPN was accompanied by an increase in blood alanine
concentration (Fig. 9.24.) to values comparable with those measured in the fed
state (400 µM) (Renold et al , 1978). In addition substrate cycles, such as the
glucose/glucose 6-phosphate and glycogen/glucose 1-phosphate cycle in the liver,
the protein/amino acid cycle in muscle, and the triacylglycerol/fatty acid cycle in
adipose tissue, may have a role during the absorptive period in amplifying the
sensitivity to small changes in concentrations of fuels and hormones (Newsholme
& Leech, 1983).
The promotion of a 'post-absorptive' period during the 12 h infusion-free
Cyclical TPN is supported by the decreases in plasma insulin and in the blood
concentrations of lactate, alanine and glucose, to levels similar to those found
after an overnight fast, such as the values measured in 21 normal volunteers
fasted overnight, used as controls in a metabolic study on glucose tolerance by
Record et al (1973). A comparison between the metabolic data published in the
latter study and those measured in this present work is shown in Table 9.9.
Cyclical Total Parenteral Nutrition, therefore, promotes a 'post-absorptive' period,
and therefore appears more physiological than Continuous Parenteral Nutrition in
terms of substrate metabolism. In addition it allows more mobility for patients
without, apparently, sacrificing efficacy.
Although Cyclical TPN patients presented a normal insulin secretory
response to the intermittent infusion of nutrients, about 30% of our patients
receiving TPN glucose-based regimens required exogenous insulin.
demonstrates the glucose intolerance and the
This
resistance to the action of insulin which can occur in parenterally fed patients
(Fig. 9.14.; Fig. .9.21. - Dudrick et al , 1970; Sanderson et al , 1974).
Table 9.9.
A comparison between blood metabolite values obtained during the 'post-
absorptive' Cyclical TPN ("OFF" or infusion free period) and those found in normal
controls after an overnight fast.
_____________________________________________________________
Results expressed as Mean±S.E.M. . * Data extracted from Record et al ,
1973.
__________________________________________________________
___
Cyclical TPN "OFF"
Overnight fast *
__________________________________________________________
___
Insulin (µUI / ml )
10.7±0.91 (50)
6.4±0.5 (21)
Lactate (µmol / ml )
0.82±0.03 (54)
0.72±0.04 (21)
Pyruvate (µmol / ml )
0.091±0.003 (52)
0.072±0.005 (21)
Ketone bodies (µmol / ml )
0.053±0.006 (56)
0.057±0.004 (21)
__________________________________________________________
____
Despite some controversy, hyperinsulinaemia has been implicated in the
development of insulin resistance in man, probably causing a defect at a postbinding site (Rizza et al , 1985). This view has been supported in a recent study
(Marangou et al , 1986) involving normal volunteers subjected to a 20 h insulin
infusion in order to promote a moderate sustained hyperinsulinaemia (30mUI / l).
This level of hyperinsulinaemia appears to be within the range of that observed in
insulin-resistant states, such as in sepsis (Fig. 3.5. - Chapter 3; Clemens et al ,
1982), injury (Black et al , 1982), chronic renal failure (DeFronzo et al , 1981),
obesity (Kolterman et al , 1980), type II diabetes (DeFronzo et al , 1982),
cirrhosis (Proietto et al , 1980), in patients with insulinoma (Bar et al , 1977), and
patients receiving Continuous TPN (Fig. 9.26. - Maini et al , 1976; Wagman et al
, 1986). This moderate hyperinsulinaemic state led to a decrease in insulin action,
despite the presence of normal monocyte-insulin receptor binding, and normal
insulin secretion Therefore, if the binding of insulin to monocytes reflects insulin
binding in key insulin-sensitive tissues, this impairment in insulin action probably
involves a post-receptor mechanism (Crettaz & Jeanrenaud, 1980). A defect at
the post-receptor level has also been suggested as the cause of insulin resistance
in injured patients (Black et al , 1982).
By contrast, Byrne et al (1981) studying parenterally fed patients with
gastrointestinal disorders, also showed no alteration in insulin binding to
circulating monocytes in patients receiving either 17 hours/day or continuous
TPN infusions, but, in a group of patients receiving Cyclical TPN (over 12 hours),
total insulin-monocyte binding was significantly diminished. This was also
accompanied by a decrease in high-affinity receptors, possibly "down-regulating"
the response to hyperinsulinaemia with each 12 h-TPN infusion. These changes in
insulin receptors (Kahn, 1980) could, therefore, help to explain why some patients
develop carbohydrate intolerance. They might also produce a protective effect
from post-infusion hypoglycaemia. The importance of the role of insulin in this
adaptation to the infusion, and cessation of nutrients is further supported by the
fact that there were no apparent changes in counter regulatory hormones in these
studies (Byrne et al , 1981). The resistance to insulin action may also be regarded
as yet another common metabolic feature between sepsis and total parenteral
nutrition.
Further investigation on the role of hyperinsulinaemia in
promoting
metabolic changes during Total Parenteral Nutrition, especially in the ill septic
patient, is therefore required. The possibility of using non-invasive techniques
may help to clarify some of these changes in the human. For example, nuclear
magnetic resonance (NMR) spectroscopy has been used as a means of identifying
hepatic steatosis in parenterally fed rats (Jacobs et al , 1986), and might become a
useful method of studying parenterally fed patients with sepsis. Indirect
calorimetry appears to be a useful tool to assess changes in hepatic metabolism in
response to TPN, such as increases in net lipogenesis (Stein, 1985). It may also be
useful in studying the net changes in fuel mobilization (Putet et al , 1984),
especially during the 'post-absorptive' period of Cyclical TPN.
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Publications arising from this Thesis
de Vasconcelos P.R.L., Kettlewell M.G.W. & Williamson D.H. (1987) Time
course of changes in hepatic metabolism in response to sepsis in the rat :
impairment of gluconeogenesis and ketogenesis
in vitro . Clinical Science , 72 , 683-691.
Abstracts
de Vasconcelos P.R.L., Williamson D.H., Ilic V., Hems R. & Kettlewell
M.G.W. (1985) Liver substrate metabolism and caloric supply during sepsis.
British Journal of Surgery , 72 , 1035.
de Vasconcelos P.R.L., Williamson D.H. & Kettlewell M.G.W. (1986) Does
sepsis impair gluconeogenesis? British Journal of Surgery , 73 , 1029.

HEPATIC METABOLISM DURING SEPSIS Paulo Roberto Leitao de

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